Plants with increased activity of a starch phosphorylating enzyme

ABSTRACT

The present invention relates to plant cells and plants, which are genetically modified, whereby the genetic modification leads to an increase in the activity of a starch-phosphorylating OK1 protein in comparison to the corresponding wild type plant cells or wild type plants that have not been genetically modified. In addition, the present invention concerns means and methods for the manufacture of such plant cells and plants. These types of plant cells and plants synthesize a modified starch. Therefore, the present invention also concerns the starches synthesized from the plant cells and plants according to the invention, methods for manufacturing these starches, and the manufacture of starch derivatives of these modified starches, as well as flours containing starches according to the invention. 
     Furthermore, the present invention also relates to nucleic acids, coding starch-phosphorylating OK1 proteins, vectors, host cells, plant cells, and plants containing such nucleic acid molecules. In addition, the present invention relates to OK1 proteins that have starch-phosphorylating activity.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a divisional patent application of U.S. patentapplication Ser. No. 13/174,228, filed Jun. 30, 2011, which is adivisional of U.S. patent application Ser. No. 12/774,420, filed May 5,2010, now U.S. Pat. No. 8,007,592, which is a divisional patentapplication of U.S. patent application Ser. No. 10/591,428, filed Sep.1, 2006, now U.S. Pat. No. 7,772,463, which is a national phaseapplication of International Patent Application No. PCT/EP2005/02449,filed Mar. 4, 2005 claims priority to EP 04090121.7, filed Mar. 29, 2004and EP 04090086.2, filed Mar. 5, 2004, the disclosures of each of whichare hereby incorporated by reference.

The present invention relates to plant cells and plants that aregenetically modified, whereby the genetic modification leads to anincrease in the activity of a starch phosphorylating OK1 protein incomparison to corresponding wild type plant cells or wild type plantsthat have not been genetically modified. The present invention alsorelates to means and methods for the manufacture of such plant cells andplants. These types of plant cells and plants synthesise a modifiedstarch. Therefore, the present invention also concerns the starchsynthesised from the plant cells and plants according to the invention,methods for the manufacture of this starch, and the manufacture ofstarch derivatives of this modified starch, as well as flours containingstarches according to the invention.

In addition, the present invention relates to nucleic acids, codingstarch phosphorylating OK1 proteins, vectors, host cells, plant cells,and plants containing such nucleic acid molecules. The present additionalso involves OK1 proteins, which exhibit starch-phosphorylatingactivity.

With regard to the increasing importance currently attributed to plantconstituents as renewable raw material sources, one of the tasks ofbiotechnological research is to endeavour to adapt these plant rawmaterials to suit the requirements of the processing industry.Furthermore, in order to enable regenerating raw materials to be used inas many areas of application as possible, it is necessary to achieve alarge variety of materials.

Polysaccharide starch is made up of chemically uniform base components,the glucose molecules, but constitutes a complex mixture of differentmolecule forms, which exhibit differences with regard to the degree ofpolymerisation and branching, and therefore differ strongly from oneanother in their physical-chemical characteristics. Discrimination ismade between amylose starch, an essentially unbranched polymer made fromalpha-1,4-glycosidically linked glucose units, and the amylopectinstarch, a branched polymer, in which the branches come about by theoccurrence of additional alpha-1,6-glycosidic links. A further essentialdifference between amylose and amylopectin lies in the molecular weight.While amylose, depending on the origin of the starch, has a molecularweight of 5×10⁵-10⁶ Da, that of the amylopectin lies between 10⁷ and 10⁸The two macromolecules can be differentiated by their molecular weightand their different physical-chemical characteristics, which can mosteasily be made visible by their different iodine bondingcharacteristics.

Amylose has long been looked upon as a linear polymer, consisting ofalpha-1,4-glycosidically linked alpha-D-glucose monomers. In more recentstudies, however, the presence of alpha-1,6-glycosidic branching points(ca. 0.1%) has been shown (Hizukuri and Takagi, Carbohydr. Res. 134,(1984), 1-10; Takeda et al., Carbohydr. Res. 132, (1984), 83-92).

The functional characteristics of starches, such as for example thesolubility, the retrogradation behaviour, the water binding capacity,the film-forming characteristics, the viscosity, the gelatinisationcharacteristics, the freezing-thawing stability, the acid stability, thegel strength and the size of the starch grain, are affected amongstother things by the amylose/amylopectin ratio, the molecular weight, thepattern of the side chain distribution, the ion concentration, the lipidand protein content, the average grain size of the starch, the grainmorphology of the starch etc. The functional characteristics of starchare also affected by the phosphate content, a non-carbon component ofstarch. Here, differentiation is made between phosphate, which is bondedcovalently in the form of monoesters to the glucose molecules of thestarch (described in the following as starch phosphate), and phosphatein the form of phospholipids associated with the starch.

The starch phosphate content varies depending on the type of plant.Therefore, certain maize mutants, for example, synthesise a starch withincreased starch phosphate content (waxy maize 0.002% and high-amylosemaize 0.013%), while conventional types of maize only have traces ofstarch phosphate. Similarly small amounts of starch phosphate are foundin wheat (0.001%), while no evidence of starch phosphate has been foundin oats and sorghum. Small amounts of starch phosphate have also beenfount in rice mutants (waxy rice 0.003%), and in conventional types, ofrice (0.013%). Significant amounts of starch phosphate have been shownin plants, which synthesise tuber or root storage starch, such astapioca (0.008%), sweet potato (0.011%), arrowroot (0.021%) or potato(0.089%) for example. The percentage values for the starch phosphatecontent quoted above refer to the dry weight of starch in each case, andhave been determined by Jane et al. (1996, Cereal Foods World 41 (11),827-832).

Starch phosphate can be present in the form of monoesters at the C-2,C-3 or C-6 position of polymerised glucose monomers (Takeda andHizukuri, 1971, Starch/Stärke 23, 267-272). The phosphate distributionof phosphate in starch synthesised by plants is generally characterisedin that approximately 30% to 40% of residual phosphate at the C-3position, and approximately 60% to 70% of the residual phosphate at theC-6 position, of the glucose molecule are covalently bonded (Blennow etal., 2000, Int. J. of Biological Macromolecules 27, 211-218). Blennow etal. (2000, Carbohydrate Polymers 41, 163-174) have determined a starchphosphate content, which is bonded in the C-6 position of the glucosemolecules, for different starches such as, for example, potato starch(between 7.8 and 33.5 nMol per mg of starch, depending on the variety),starch from different Curcuma species (between 1.8 and 63 nMol per mg),tapioca starch (2.5 nMol per mg of starch), rice starch (1.0 nMol per mgof starch), mung bean starch (3.5 nMol per mg of starch) and sorghumstarch (0.9 nMol per mg of starch). These authors have been unable toshow any starch phosphate bonded at the C-6 position in barley starchand starches from different waxy mutants of maize. Up to now, it has notbeen possible to establish a connection between the genotype of a plantand the starch phosphate content (Jane et al., 1996, Cereal Foods World41 (11), 827-832). It is therefore currently not possible to affect thestarch phosphate content in plants by means of breeding measures.

Previously, only one protein has been described, which facilitates theintroduction of covalent bonds of phosphate residues to the glucosemolecules of starch. This protein has the enzymatic activity of analpha-glucan-water dikinase (GWD, E.C.: 2.7.9.4) (Ritte et al., 2002,PNAS 99, 7166-7171), is frequently described in the literature as R1,and is bonded to the starch grains of the storage starch in potatotubers (Lorberth et al., 1998, Nature Biotechnology 16, 473-477). In thereaction catalysed by R1, the educts alpha-1,4-glucan (starch),adenosintriphosphate (ATP) and water are converted to the productsglucan-phosphate (starch phosphate), monophosphate and adenosinemonophosphate. In doing so, the residual gamma phosphate of the ATP istransferred to water, and the residual beta phosphate of the ATP istransferred to the glucan (starch). R1 transfers the residual betaphosphate of ATP to the C-6 and the C-3 position of the glucosemolecules of alpha-1,4-glucans in vitro. The ratio of C-6 phosphate toC-3 phosphate, which is obtained in the in vitro reaction, is the sameas the ratio, which is present in starch isolated from plants (Ritte etal., 2002, PNAS 99, 7166-7171). As about 70% of the starch phosphatepresent in potato starch is bonded to the glucose monomers of the starchin the C-6 position and about 30% in the C-3 position, this means thatR1 preferably phosphorylates the C-6 position of the glucose molecules.Furthermore, it has been shown that by the use of amylopectin frommaize, amongst other things, R1 can phosphorylate alpha-1,4-glucans,which do not yet contain covalently bonded phosphate (Ritte et al.,2002, PNAS 99, 7166-7171), i.e. R1 is able to introduce phosphate denovo into alpha-1,4-glucans.

Nucleic acid sequences, and the amino acid sequences corresponding tothem, coding an R1 protein, are described from different species, suchas, for example, potato (WO 97 11188, GenBank Acc.: AY027522, Y09533),wheat (WO 00 77229, U.S. Pat. No. 6,462,256, GenBank Acc.: AAN93923,GenBank Acc.: AR236165), rice (GenBank Acc.: AAR61445, GenBank Acc.:AR400814), maize (GenBank Acc.: AAR61444, GenBank Acc.: AR400813), soyabean (GenBank Acc.: AAR61446, GenBank Acc.: AR400815), citrus (GenBankAcc.: AY094062) and Arabidopsis (GenBank Acc.: AF312027).

Wheat plants, which exhibit increased activity of an R1 protein due tooverexpression of an R1 potato gene, are described in WO 02 34923. Theseplants synthesise a starch with significant quantities of starchphosphate at the C-6 position of the glucose molecules in comparison tocorresponding wild type plants, in which no starch phosphate could bedetected.

Further proteins, which catalyse a reaction, which introduce covalentlybonded phosphate groups into the starch, have not previously beendescribed. Enzymes, which preferably introduce phosphate groups in theC-3 position and/or the C-2 position of the glucose molecules of starch,are also unknown. Apart from the increase of the starch phosphatecontent in plants, there are therefore also no available ways ofspecifically influencing the phosphorylation of starch in plants, ofmodifying the phosphate distribution within the starch synthesised byplants and/or of further increasing the starch phosphate content.

The object of the present invention is therefore based on providingmodified starches with increased phosphate content and/or modifiedphosphate distribution, as well as plant cells and/or plants, whichsynthesise such a modified starch, as well as means and methods forproducing said plants and/or plant cells.

This problem is solved by the embodiments described in the claims.

The present invention therefore relates to genetically modified plantcells and genetically modified plants, characterised in that the plantcells or plants have an increased activity of at least one OK1 proteinin comparison with corresponding wild type plant cells or wild typeplants that have not been genetically modified.

A first aspect of the present invention relates to a plant cell orplant, which is genetically modified, wherein the genetic modificationleads to an increase in the activity of at least one OK1 protein incomparison with corresponding wild type plant cells or wild type plantsthat have not been genetically modified.

At the same time, the genetic modification can be any geneticmodification, which leads to an increase in the activity of at least oneOK1 protein in comparison with corresponding wild type plant cells orwild type plants that have not been genetically modified.

In conjunction with the present invention, the term “wild type plantcell” means that the plant cells concerned were used as startingmaterial for the manufacture of the plant cells according to theinvention, i.e. their genetic information, apart from the introducedgenetic modification, corresponds to that of a plant cell according tothe invention.

In conjunction with the present invention, the term “wild type plant”means that the plants concerned were used as starting material for themanufacture of the plants according to the invention, i.e. their geneticinformation, apart from the introduced genetic modification, correspondsto that of a plant according to the invention.

In conjunction with the present invention, the term “corresponding”means that, in the comparison of several objects, the objects concernedthat are compared with one another have been kept under the sameconditions. In conjunction with the present invention, the term“corresponding” in conjunction with wild type plant cell or wild typeplant means that the plant cells or plants, which are compared with oneanother, have been raised under the same cultivation conditions and thatthey have the same (cultivation) age.

The term, “increased activity of at least one OK1 protein” within theframework of the present invention means an increase in the expressionof endogenous genes, which code the OK1 proteins, and/or an increase inthe quantity of OK1 proteins in the cells, and/or an increase in theenzymatic activity of OK1 proteins in the cells.

The increase in the expression can be determined by measuring thequantity of OK1 proteins coding transcripts, for example; e.g. by way ofNorthern Blot analysis or RT-PCR. An increase preferably means anincrease in the quantity of transcripts of at least 50%, preferably atleast 70%, more preferably at least 85%, and most preferably at least100%, in comparison to corresponding cells that have not beengenetically modified. An increase in the quantity of transcripts codingan OK1 protein also means that plants or plant cells, which do notexhibit any detectable quantities of transcripts coding an OK1 protein,show detectable quantities of transcripts coding an OK1 proteinfollowing genetic modification according to the invention.

The increase in the amount of protein of an OK1 protein, which resultsin increased activity of this protein in the plant cells concerned, can,for example, be determined by immunological methods such as Western blotanalysis, ELISA (Enzyme Linked Immuno Sorbent Assay) or RIA (RadioImmune Assay). Here, an increase preferably means an increase in theamount of OK1 protein in comparison with corresponding plant cells thathave not been genetically modified by at least 50%, in particular by atleast 70%, preferably by at least 85% and particularly preferably by atleast 100%. An increase in the amount of OK1 protein also means thatplants or plant cells that do not have any detectable OK1 proteinactivity exhibit a detectable quantity of OK1 protein following geneticmodification according to the invention.

Methods for manufacturing antibodies, which react specifically with acertain protein, i.e. which bond specifically to said protein, are knownto the person skilled in the art (see, for example, Lottspeich andZorbas (Eds.), 1998, Bioanalytik, Spektrum akad, Verlag, Heidelberg,Berlin, ISBN 3-8274-0041-4). The manufacture of such antibodies isoffered by some companies (e.g. Eurogentec, Belgium) as a contractservice. A possible way of manufacturing antibodies, which specificallyreact with an OK1 protein, is described below (see Example 10).

Within the framework of the present invention, the term “OK1 protein” isto be understood to mean a protein, which transfers a phosphate residueof ATP onto already phosphorylated starch (P-starch). Starches isolatedfrom leaves of an Arabisopsis thaliana sex1-3 mutant have no detectableamounts of covalently bonded phosphate residues and are notphosphorylated by an OK1 protein, i.e. an OK1 protein according to theinvention requires already phosphorylated starch as a substrate fortransferring further phosphate residues.

Preferably, the residual beta phosphate of the ATP is transferred froman OK1 protein to the starch, and the residual gamma phosphate of theATP is transferred to water. A further reaction product produced by aphosphorylating reaction of P-starch carried out using an OK1 protein isAMP (adenosine monophosphate). An OK1 protein is therefore described as[phosphorylated-alpha-glucan]-water-dikinase ([P-glucan]-water-dikinase)or as [phosphorylated-starch]-water-dikinase.

Preferably, an additional phosphate monoester bond is produced in theC-6 position and/or in the C-3 position of a glucose molecule of theP-starch, which is phosphorylated by an OK1 protein. In thephosphorylation of P-starch catalysed by an OK1 protein, it isparticularly preferred if more additional phosphate monoester bonds areproduced in the C-3 position in comparison with phosphate monoesterbonds in the C-6 position of the glucose molecules of the P-starchconcerned.

Amino acid sequences, which code OK1 proteins, contain aphosphohistidine domain. Phosphohistidine domains are described, forexample, by Tien-Shin Yu et al. (2001, Plant Cell 13, 1907-1918).Phosphohistidine domains of OK1 proteins coding amino acids preferablycontain two histidines.

In the catalysis of a phosphorylating reaction of a P-starch by means ofan OK1 protein, a phosphorylated OK1 protein is produced as anintermediate product, in which a phosphate residue of ATP is covalentlybonded to an amino acid of the OK1 protein. The intermediate product isproduced by autophosphorylation of the OK1 protein, i.e. the OK1 proteinitself catalyses the reaction, which leads to the intermediate product.Preferably, a histidine residue of the amino acid sequence coding an OK1protein is phosphorylated as a result of the autophosphorylationprocess, particularly preferably a histidine residue, which is part of aphosphohistidine domain.

Furthermore, OK1 proteins according to the invention have an increasedbonding activity to P-starch in comparison with non-phosphorylatedstarches.

As no enzymes have previously been described, which require P-starch asa substrate in order to phosphorylate them further, it has alsopreviously not been possible to increase the starch phosphate content ofalready phosphorylated starch in plants above a certain quantity. Thisis now possible with the utilisation of a protein according to theinvention or a nucleic acid molecule according to the invention for thegenetic modification of plants. The clarification of the function of anOK1 protein, and thus the provision of an OK1 protein, leads to the factthat plants can now be genetically modified in such a way that theysynthesise a starch with modified characteristics. The modification ofthe phosphate distribution in starch synthesised by plants waspreviously not possible due to the lack of available means. Due to theprovision by the present invention of proteins and nucleic acidsaccording to the invention, it is now also possible to modify thephosphate ratio in native starches.

In conjunction with the present invention, the term “increased bondingactivity” is to be understood to mean an increased affinity of a proteinto a first substrate in comparison with a second substrate. That is tosay, the amount of protein, which, under the same incubation conditions,bonds to a first substrate to a greater extent in comparison with asecond substrate, exhibits increased bonding activity to the firstsubstrate.

In conjunction with the present invention, the term “starch phosphate”is to be understood to mean phosphate groups covalently bonded to theglucose molecules of starch.

In conjunction with the present invention, the term “non-phosphorylatedstarch” is to be understood to mean a starch, which does not contain anydetectable amounts of starch phosphate. Different methods of determiningthe amount of starch phosphate are described. Preferably, the method ofdetermining the amount of starch phosphate described by Ritte et al.(2000, Starch/Stärke 52, 179-185) can be used. Particularly preferably,the determination of the amount of starch phosphate by means of ³¹P-NMRis carried out according to the method described by Kasemusuwan and Jane(1996, Cereal Chemistry 73, 702-707).

In conjunction with the present invention, the term “phosphorylatedstarch” or “P-starch” is to be understood to mean a starch, whichcontains starch phosphate.

The activity of an OK1 protein can be demonstrated, for example, by invitro incubation of an OK1 protein using ATP, which contains a phosphateresidue labeled in the beta position (labeled ATP). Preferably ATP isused, in which the phosphate residue is specifically labeled in the betaposition, i.e. in which only the phosphate residue in the beta positionhas a marking. Preferably radioactively labeled ATP, particularlypreferably ATP, in which the phosphate residue is specificallyradioactively labeled in the beta position, and especially preferablyATP, in which the phosphate residue in the beta position is specificallylabeled with ³³P, is used. If an OK1 protein with labeled ATP andstarches, which are not phosphorylated, are incubated, no phosphate istransferred to the starch due to OK1. Preferably, leaf starch ofArabidopsis thaliana mutant sex1-3 (Tien-Shin Yu et al., 2001, PlantCell 13, 1907-1918) is used.

If, on the other hand, an OK1 protein with P-starch is incubated in thepresence of labeled ATP, then labeled phosphate covalently bonded to theP-starch can subsequently be shown. Preferably, starch from leaves ofArabidopsis thaliana, particularly preferably starch from Arabidopsisthaliana sex1-3 mutants enzymatically phosphorylated by means of an R1protein (Ritte et al., 2002, PNAS 99, 7166-7171) is used.

Labeled phosphate residues, which have been incorporated in P-starch dueto an OK1 protein, e.g. by separating the labeled P-starch (e.g. byprecipitation with ethanol, filtration, chromatographic methods etc.)from the rest of the reaction mixture and subsequently detecting thelabeled phosphate residue in the P-starch fraction, can be shown. At thesame time, the labeled phosphate residues bonded in the P-starchfraction can be demonstrated, for example, by determining the amount ofradioactivity present in the P-starch fraction (e.g. by means ofscintillation counters). Possible methods for demonstrating a protein,which requires P-starch as a substrate for a phosphorylating reaction,are described below under General Methods, Item 11 and in Example 6.

Which positions of the carbon atoms (C-2, C-3 or C-6) of the glucosemonomers in P-starch are preferably phosphorylated by an OK1 protein canbe determined, for example, by analysing the P-starches phosphorylatedby a protein, as described by Ritte et al. (2002, PNAS 99, 7166-7171).For this purpose, a P-starch phosphorylated by a protein is hydrolysedusing an acid, and subsequently analysed by means of anion exchangechromatography.

Preferably, the P-starch phosphorylated by an OK1 protein is analysed bymeans of NMR in order to establish which positions of the carbon atoms(C-2, C-3 or C-6) of the glucose monomers in the P-starch arephosphorylated. A particularly preferred method for identifying theC-atom positions of a glucose molecule of a starch, which arephosphorylated by a reaction catalysed by an OK1 protein, is describedbelow under General Methods, Item 13.

A phosphorylated protein, which is produced as an intermediate productin the phosphorylation of P-starch facilitated by an OK1 protein, can bedemonstrated as described, for example, by Ritte et al. (2002, PNAS 99,7166-7171) for an R1 protein.

To demonstrate the presence of an autophosphorylated intermediateproduct, an OK1 protein is first incubated in the absence of starch withlabeled ATP, preferably with ATP specifically labeled in the betaphosphate position, particularly preferably with ATP specificallylabeled with ³³P in the beta phosphate position. In parallel with this,a reaction preparation 2, which instead of labeled ATP containscorresponding amounts of non-labeled ATP however, is incubated underotherwise identical conditions. Subsequently, an excess of unlabeled ATPis added to reaction mixture 1 and a mixture of unlabeled ATP andlabeled ATP (the same quantity of labeled ATP as was used previously inreaction mixture 1, and the same quantity of the excess of unlabeled ATPthat was added to reaction mixture 1) is added to reaction mixture 2,and this is further incubated before adding P-starch to Part A ofreaction mixture 1 (Part 1A) and to Part A of reaction mixture 2 (Part2A). The reaction in the remaining Part 1B and Part 2B of the reactionmixture is stopped by denaturing the protein. Part B of the reactionmixture can be stopped by the methods known to the person skilled in theart, which lead to the denaturing of proteins, preferably by addingsodium lauryl sulphate (SDS). Part 1A and Part 2A of the reactionmixture are incubated for at least a further 10 minutes before thesereactions are also stopped. The starch present in Part A and Part B ofthe respective reaction mixture is separated from the remainder of thereaction mixture. If the respective starch is separated bycentrifugation, for example, then, on completion of centrifugation, thestarch of the respective Part A or Part B of the reaction mixture is tobe found in the sedimented pellet, and the proteins in the respectivereaction mixture are to be found in the supernatant of the respectivecentrifugation. The supernatant of Part 1A or 2A respectively and Part1B or 2B respectively of the reaction mixture can subsequently beanalysed by denaturing acrylamide gel electrophoresis, for example,followed by autoradiography of the acrylamide gel obtained. To quantifythe amount of radioactively labeled proteins, which have been separatedby means of acrylamide gel electrophoresis, the so-called“phospho-imaging” method, for example, known to the person skilled inthe art, can be used. If the autoradiography or the analysis by means ofthe “phospho-imager” of proteins in the centrifugation supernatant ofPart B of reaction mixture 1 shows a significantly increased signalcompared with the centrifugation excess of Part A of reaction mixture 1,then this shows that a protein facilitating a phosphorylation of starchoccurs as an autophosphorylated intermediate product. Parts A and B ofreaction mixture 2 serve as a control and should therefore not exhibit asignificantly increased signal in the centrifugation supernatant in theautoradiography or in the analysis by means of the “phospho-imager”.

In addition, the starch of the respective Part A of reaction mixture 1and 2 remaining in the respective sedimented pellet can be investigated,if necessary after subsequent washing of the respective starches, forthe presence of starch phosphate, which has a mark corresponding to thelabeled ATP used. If the starches of Part A of reaction mixture 1contain labeled phosphate residues, and if the autoradiography of thecentrifugation supernatant of Part B of reaction mixture 1 shows asignificantly increased signal in the autoradiography compared with thecentrifugation supernatant of Part A of reaction mixture 1, then thisshows that a phosphorylation of starch-facilitating protein is presentas an autophosphorylated intermediate product. Parts A and B of reactionmixture 2 serve as a control and should therefore not exhibit asignificantly increased signal for alpha-1,4-glucans labeled with ³³P inthe sedimented pellet containing alpha-1,4-glucans. Possible methods fordemonstrating a phosphorylated OK1 protein intermediate product aredescribed below under General Methods, Item 12 and in Example 7.

That an OK1 protein has an increased bonding activity to a P-starchcompared with non-phosphorylated starch can be demonstrated byincubating the OK1 protein with P-starch and non-phosphorylated starchin separate preparations.

All non-phosphorylated starches are basically suitable for incubatingOK1 proteins with non-phosphorylated starch. Preferably, anon-phosphorylated plant starch, particularly preferably wheat starch,and especially preferably granular leaf starch of an Arabidopsisthaliana mutant sex1-3 is used.

Methods for isolating starch from plants, for example, are known to theperson skilled in the art. All methods known to the person skilled inthe art are basically suitable for isolating non-phosphorylated starchfrom appropriate plant species. Preferably, the methods for isolatingnon-phosphorylated alpha-1,4-glucans described below are used (seeGeneral Methods Item 2).

All starches, which contain starch phosphate, are basically suitable forincubating OK1 proteins with P-starch. Chemically phosphorylatedstarches can also be used for this purpose. Preferably, P-starches areused for the incubation with OK1 proteins, particularly preferably aretrospectively enzymatically phosphorylated plant starch, especiallypreferably a retrospectively enzymatically phosphorylated plant granularstarch, which has been isolated from a sex-1 mutant of Arabidopsisthaliana.

To demonstrate an increased bonding activity of OK1 proteins to P-starchcompared with non-phosphorylated starch, OK1 proteins are incubated inseparate preparations with P-starch (Preparation A) and withnon-phosphorylated starch (Preparation B). After successful incubation,the proteins, which are not bonded to the relevant starches ofpreparations A and B, are separated from the starches and the proteinsto which they are bonded. The bond between the proteins and the P-starchin Preparation A and the bond between the proteins andnon-phosphorylated starch in Preparation B are subsequently removed,i.e. the respective proteins are dissolved. The dissolved proteins ofPreparation A and Preparation B can then be separated from the starchesconcerned, which are present in the respective preparations. Followingthis, the isolated P-starch bonding proteins of Preparation A and theisolated non-phosphorylated starch bonding proteins of Preparation B canbe separated with the help of methods known to the person skilled in theart such as, for example, gel filtration, chromatographic methods,electrophoresis, SDS acrylamide gel electrophoresis etc. By comparingthe amounts of separated proteins of Preparation A with the amounts ofcorresponding separated proteins of Preparation B, it can be determinedwhether a protein has an increased bonding activity with respect toP-starch compared with non-phosphorylated starch. Methods, which can beused to demonstrate a preferred bonding of proteins to P-starch comparedwith non-phosphorylated starch, are described below in (General Methods,Item 8 and Example 1).

The amino acid sequence shown in SEQ ID NO 2 codes an OK1 protein fromArabidopsis thaliana and the amino acid sequence shown under SEQ ID NO 4codes an OK1 protein from Oryza sativa.

In a further embodiment of the present invention, amino acid sequencescoding an OK1 protein have an identity of at least 60% with the sequencespecified in SEQ ID NO 2 or SEQ ID NO 4, in particular of at least 70%,preferably of at least 80% and particularly preferably of at least 90%and especially preferably of at least 95%.

In a further embodiment of the present invention, the OK1 proteinexhibits a phosphohistidine domain (Tien-Shin Yu et al., 2001, PlantCell 13, 1907-1918). Amino acid sequences coding OK1 proteins contain aphosphohistidine domain, which exhibits an identity of at least 50%, inparticular of at least 60%, preferably of at least 70%, particularlypreferably of at least 80%, and more particularly preferably of at least90% of the amino acid sequence of the phosphohistidine domain of the OK1protein from Arabidopsis thaliana and Oryza sativa, specified under SEQID NO 5. The phosphohistidine domain preferably contains two histidinesresidues.

A further embodiment of the present invention relates to a geneticallymodified plant cell according to the invention or a genetically modifiedplant according to the invention, wherein the genetic modificationconsists in the introduction of at least one foreign nucleic acidmolecule into the genome of the plant.

In this context, the term “genetic modification” means the introductionof homologous and/or heterologous foreign nucleic acid molecules intothe genome of a plant cell or into the genome of a plant, wherein saidintroduction of these molecules leads to an increase in the activity ofan OK1 protein.

The plant cells according to the invention or plants according to theinvention are modified with regard to their genetic information by theintroduction of a foreign nucleic acid molecule. The presence or theexpression of the foreign nucleic acid molecule leads to a phenotypicchange. Here, “phenotypic” change means preferably a measurable changeof one or more functions of the cells. For example, the geneticallymodified plant cells according to the invention and the geneticallymodified plants according to the invention exhibit an increase in theactivity of an OK1 protein due to the presence of or in the expressionof the introduced nucleic acid molecule.

In conjunction with the present invention, the term “foreign nucleicacid molecule” is understood to mean such a molecule that either doesnot occur naturally in the corresponding wild type plant cells, or thatdoes not occur naturally in the concrete spatial arrangement in wildtype plant cells, or that is localised at a place in the genome of theplant cell at which it does not occur naturally in wild type plantcells. Preferably, the foreign nucleic acid molecule is a recombinantmolecule, which consists of different elements, the combination orspecific spatial arrangement of which does not occur naturally in plantcells.

In principle, the foreign nucleic acid molecule can be any nucleic acidmolecule, which causes an increase in the activity of an OK1 protein inthe plant cell or plant.

In conjunction with the present invention, the term “genome” is to beunderstood to mean the totality of the genetic material present in aplant cell. It is known to the person skilled in the art that, inaddition to the cell nucleus, other compartments (e.g. plastids,mitochondria) also contain genetic material.

In a further embodiment, the plant cells according to the invention andthe plants according to the invention are characterised in that theforeign nucleic acid molecule codes an OK1 protein, preferably an OK1protein from Arabidopsis thaliana or an OK1 protein from Oryza sativa.

In a further embodiment, the foreign nucleic acid molecule codes an OK1protein with the amino acid sequence specified in SEQ ID NO 2 or SEQ IDNO 4.

A large number of techniques are available for the introduction of DNAinto a plant host cell. These techniques include the transformation ofplant cells with T-DNA using Agrobacterium tumefaciens or Agrobacteriumrhizogenes as the transformation medium, the fusion of protoplasts,injection, the electroporation of DNA, the introduction of DNA by meansof the biolistic approach as well as other possibilities. The use ofagrobacteria-mediated transformation of plant cells has been intensivelyinvestigated and adequately described in EP 120516; Hoekema, Ind.: TheBinary Plant Vector System Offsetdrukkerij Kanters B. V., Alblasserdam(1985), Chapter V; Fraley et al., Crit. Rev. Plant Sci. 4, 1-46 and byAn et al. EMBO J. 4, (1985), 277-287. For the potato transformation, seeRocha-Sosa et al., EMBO J. 8, (1989), 29-33, for example.

The transformation of monocotyledonous plants by means of vectors basedon Agrobacterium transformation has also been described (Chan et al.,Plant Mol. Biol. 22, (1993), 491-506; Hiei et al., Plant J. 6, (1994)271-282; Deng et al, Science in China 33, (1990), 28-34; Wilmink et al.,Plant Cell Reports 11, (1992), 76-80; May et al., Bio/Technology 13,(1995), 486-492; Conner and Domisse, Int. J. Plant Sci. 153 (1992),550-555; Ritchie et al, Transgenic Res. 2, (1993), 252-265). Analternative system to the transformation of monocotyledonous plants istransformation by means of the biolistic approach (Wan and Lemaux, PlantPhysiol. 104, (1994), 37-48; Vasil et al., Bio/Technology 11 (1993),1553-1558; Ritala et al., Plant Mol. Biol. 24, (1994), 317-325; Spenceret al., Theor. Appl. Genet. 79, (1990), 625-631), protoplasttransformation, electroporation of partially permeabilised cells and theintroduction of DNA by means of glass fibres. In particular, thetransformation of maize has been described in the literature many times(cf. e.g. WO95/06128, EP0513849, EP0465875, EP0292435; Fromm et al.,Biotechnology 8, (1990), 833-844; Gordon-Kamm et al., Plant Cell 2,(1990), 603-618; Koziel et al., Biotechnology 11 (1993), 194-200; Morocet al., Theor. Appl. Genet. 80, (1990), 721-726).

The successful transformation of other types of cereal has also alreadybeen described, for example for barley (Wan and Lemaux, see above;Ritala et al., see above; Krens et al., Nature 296, (1982), 72-74) andfor wheat (Nehra et al., Plant J. 5, (1994), 285-297; Becker et al.,1994, Plant Journal 5, 299-307). All the above methods are suitablewithin the framework of the present invention.

Amongst other things, plant cells and plants, which have beengenetically modified by the introduction of an OK1 protein, can bedifferentiated from wild type plant cells and wild type plantsrespectively in that they contain a foreign nucleic acid molecule, whichdoes not occur naturally in wild type plant cells or wild type plants,or in that such a molecule is present integrated at a place in thegenome of the plant cell according to the invention or in the genome ofthe plant according to the invention at which it does not occur in wildtype plant cells or wild type plants, i.e. in a different genomicenvironment. Furthermore, plant cells according to the invention andplants according to the invention of this type differ from wild typeplant cells and wild type plants respectively in that they contain atleast one copy of the foreign nucleic acid molecule stably integratedwithin their genome, possibly in addition to naturally occurring copiesof such a molecule in the wild type plant cells or wild type plants. Ifthe foreign nucleic acid molecule(s) introduced into the plant cellsaccording to the invention or into the plants according to the inventionis (are) additional copies of molecules already occurring naturally inthe wild type plant cells or wild type plants respectively, then theplant cells according to the invention and the plants according to theinvention can be differentiated from wild type plant cells or wild typeplants respectively in particular in that this additional copy or theseadditional copies is (are) localised at places in the genome at which itdoes not occur (or they do not occur) in wild type plant cells or wildtype plants. This can be verified, for example, with the help of aSouthern blot analysis.

Furthermore, the plant cells according to the invention and the plantsaccording to the invention can preferably be differentiated from wildtype plant cells or wild type plants respectively by at least one of thefollowing characteristics: If the foreign nucleic acid molecule that hasbeen introduced is heterologous with respect to the plant cell or plant,then the plant cells according to the invention or plants according tothe invention have transcripts of the introduced nucleic acid molecules.These can be verified, for example, by Northern blot analysis or byRT-PCR (Reverse Transcription Polymerase Chain Reaction). Plant cellsaccording to the invention and plants according to the invention, whichexpress an antisense and/or an RNAi transcript, can be verified, forexample, with the help of specific nucleic acid probes, which arecomplimentary to the RNA (occurring naturally in the plant cell), whichis coding for the protein. Preferably, the plant cells according to theinvention and the plants according to the invention contain a protein,which is coded by an introduced nucleic acid molecule. This can bedemonstrated by immunological methods, for example, in particular by aWestern blot analysis.

If the foreign nucleic acid molecule that has been introduced ishomologous with respect to the plant cell or plant, the plant cellsaccording to the invention or plants according to the invention can bedifferentiated from wild type plant cells or wild type plantsrespectively due to the additional expression of the introduced foreignnucleic acid molecule, for example. The plant cells according to theinvention and the plants according to the invention preferably containtranscripts of the foreign nucleic acid molecules. This can bedemonstrated by Northern blot analysis, for example, or with the help ofso-called quantitative PCR.

In a further embodiment, the plant cells according to the invention andthe plants according to the invention are transgenic plant cells ortransgenic plants respectively.

In a further embodiment, the present invention relates to plant cellsaccording to the invention and plants according to the invention whereinthe foreign nucleic acid molecule is chosen from the group consistingof:

-   a) Nucleic acid molecules, which code a protein with the amino acid    sequence given under SEQ ID NO 2 or SEQ ID NO 4;-   b) Nucleic acid molecules, which code a protein, which includes the    amino acid sequence, which is coded by the insertion in plasmid    A.t.-OK1-pGEM or the insertion in plasmid pMI50;-   c) Nucleic acid molecules, which code a protein, the sequence of    which has an identity of at least 60% with the amino acid sequence    given under SEQ ID NO 2 or SEQ ID NO 4;-   d) Nucleic acid molecules, which code a protein, the sequence of    which has an identity of at least 60% with the amino acid sequence,    which is coded by the coding region of the insertion in plasmid    A.t.-OK1-pGEM or by the coding region of the insertion in plasmid    pMI50;-   e) Nucleic acid molecules, which include the nucleotide sequence    shown under SEQ ID NO 1 or SEQ ID NO 3 or a complimentary sequence;-   f) Nucleic acid molecules, which include the nucleotide sequence of    the insertion contained in plasmid A.t.-OK1-pGEM or plasmid pMI50;-   g) Nucleic acid molecules, which have an identity of at least 60%    with the nucleic acid sequences described under a), b), e) or f);-   h) Nucleic acid molecules, which hybridise with at least one strand    of the nucleic acid molecules described under a), b), d), e) or f)    under stringent conditions;-   i) Nucleic acid molecules, the nucleotide sequence of which deviates    from the sequence of the nucleic acid molecules identified under a),    b), e) or f) due to the degeneration of the genetic code; and-   j) Nucleic acid molecules, which represent fragments, allelic    variants and/or derivatives of the nucleic acid molecules identified    under a), b), c), d), e), f), g), h) or i).

The amino acid sequence shown in SEQ ID NO 2 codes an OK1 protein fromArabidopsis thaliana and the amino acid sequence shown in SEQ ID NO 4codes an OK1 protein from Oryza sativa.

The proteins coded from the different varieties of nucleic acidmolecules according to the invention have certain commoncharacteristics. These can include, for example, biological activity,molecular weight, immunological reactivity, conformation etc, as well asphysical characteristics such as, for example, the running behaviour ingel electrophoresis, chromatographic behaviour, sedimentationcoefficients, solubility, spectroscopic characteristics, stability;optimum pH, optimum temperature etc.

The molecular weight of the OK1 protein from Arabidopsis thalianaderived from the amino acid sequence shown under SEQ ID NO 2 is ca. 131kDa and the molecular weight of the OK1 protein from Oryza sativaderived from the amino acid sequence shown under SEQ ID NO 4 is ca. 132kDa. The derived molecular weight of a protein according to theinvention therefore preferably lies in the range from 120 kDa to 145kDa, preferably in the range from 120 kDa to 140 kDa, particularlypreferably from 125 kDa to 140 kDa and especially preferably from 130kDa to 135 kDa.

The amino acid sequences shown in SEQ ID NO 2 and SEQ ID NO 4 coding OK1proteins from Arabidopsis thaliana and Oryza sativa respectively eachcontain a phosphohistidine domain. Preferably, an OK1 protein accordingto the invention therefore contains a phosphohistidine domain, which hasan identity of at least 50%, preferably of at least 60%, particularlypreferably of at least 80% and especially preferably of 90% with thephosphohistidine domain shown under SEQ ID NO 5.

The present invention relates to nucleic acid molecules, which code aprotein with the enzymatic activity according to the invention of an OK1protein, wherein the coded OK1 protein has an identity of at least 70%,preferably of at least 80%, particularly preferably of at least 90% andespecially preferably of 95% with the amino acid sequence specifiedunder SEQ ID NO 2 or SEQ ID NO 4.

A plasmid (A.t.-OK1-pGEM) containing a cDNA which codes for a proteinaccording to the invention (A.t.-OK1) from Arabidopsis thaliana wasdeposited on 8 Mar. 2004 under the number DSM16264 and a plasmid (pM150)containing a cDNA which codes for further protein according to theinvention (O.s.-OK1) from Oryza sativa was deposited on 24 Mar. 2004under the number DSM16302 under the Budapest Treaty at the GermanCollection of Microorganisms and Cell Cultures GmbH, Mascheroder Weg 1b,38124 Braunschweig, Germany.

The amino acid sequence shown in SEQ ID NO 2 can be derived from thecoding region of the cDNA sequence integrated in plasmid A.t.-OK1-pGEMand codes for an OK1 protein from Arabidopsis thaliana. The amino acidsequence shown in SEQ ID NO 4 can be derived from the coding region ofthe cDNA sequence integrated in plasmid pMI50 and codes for an OK1protein from Oryza sativa. The present invention therefore also relatesto nucleic acid molecules, which code a protein with the enzymaticactivity of an OK1 protein, which includes the amino acid sequence,which is coded by the insertion in plasmid A.t.-OK1-pGEM or by theinsertion in plasmid pMI50, wherein the coded protein has an identity ofat least 70%, preferably of at least 80%, particularly preferably of atleast 90% and especially preferably of 95% with the amino acid sequence,which can be derived from the insertion in A.t.-OK1-pGEM or pMI50.

The nucleic acid sequence shown in SEQ ID NO 1 is a cDNA sequence, whichincludes the coding region for an OK1 protein from Arabidopsis thalianaand the nucleic acid sequence shown in SEQ ID NO 3 is a cDNA sequence,which includes the coding region for an OK1 protein from Oryza sativa.

The present invention therefore also relates to nucleic acid molecules,which code an OK1 protein and the coding region of the nucleotidesequences shown under SEQ ID NO 1 or SEQ ID NO 3 or sequences, which arecomplimentary thereto, nucleic acid molecules, which include the codingregion of the nucleotide sequence of the insertion contained in plasmidA.t.-OK1-pGEM or in plasmid pMI50 and nucleic acid molecules, which havean identity of at least 70%, preferably of at least 80%, particularlypreferably of at least 90% and especially preferably of at least 95%with the said nucleic acid molecules.

With the help of the sequence information of nucleic acid moleculesaccording to the invention or with the help of a nucleic acid moleculeaccording to the invention, it is possible for the person skilled in theart to isolate homologous sequences from other plant species, preferablyfrom starch-storing plants, preferably from plant species of the genusOryza, in particular Oryza sativa or from Arabidopsis thaliana. This canbe carried out, for example, with the help of conventional methods suchas the examination of cDNA or genomic libraries with suitablehybridisation samples. The person skilled in the art knows thathomologous sequences can also be isolated with the help of (degenerated)oligonucleotides and the use of PCR-based methods.

The examination of databases, such as are made available, for example,by EMBL (http://www.ebi.ac.uk/Tools/index.htm) or NCBI (National Centerfor Biotechnology Information, http://www.ncbi.nlm.nih.gov/), can alsobe used for identifying homologous sequences, which code for OK1protein. In this case, one or more sequences are specified as aso-called query. This query sequence is then compared by means ofstatistical computer programs with sequences, which are contained in theselected databases. Such database queries (e.g. blast or fasta searches)are known to the person skilled in the art and can be carried out byvarious providers.

If such a database query is carried out, e.g. at the NCBI (NationalCenter for Biotechnology Information, http://www.ncbi.nlm.nih.gov/),then the standard settings, which are specified for the particularcomparison inquiry, should be used. For protein sequence comparisons(blastp), these are the following settings: Limit entrez=not activated;Filter=low complexity activated; Expect value=10; word size=3;Matrix=BLOSUM62; Gap costs: Existence=11, Extension=1.

For nucleic acid sequence comparisons (blastn), the following parametersmust be set: Limit entrez=not activated; Filter=low complexityactivated; Expect value=10; word size=11.

With such a database search, the sequences described in the presentinvention can be used as a query sequence in order to identify furthernucleic acid molecules and/or proteins, which code an OK1 protein.

With the help of the described methods, it is also possible to identifyand/or isolate nucleic acid molecules according to the invention, whichhybridise with the sequence specified under SEQ ID NO 1 or under SEQ IDNO 3 and which code an OK1 protein.

Within the framework of the present invention, the term “hybridising”means hybridisation under conventional hybridisation conditions,preferably under stringent conditions such as, for example, aredescribed in Sambrock et al., Molecular Cloning, A Laboratory Manual,3rd edition (2001) Cold Spring Harbor Laboratory Press, Cold SpringHarbor, N.Y. ISBN: 0879695773, Ausubel et al., Short Protocols inMolecular Biology, John Wiley & Sons; 5th edition (2002), ISBN:0471250929). Particularly preferably, “hybridising” means hybridisationunder the following conditions:

Hybridisation buffer:

-   -   2×SSC; 10×Denhardt solution (Ficoll 400+PEG+BSA; Ratio 1:1:1);        0.1% SDS; 5 mM EDTA; 50 mM Na2HPO4; 250 μg/ml herring sperm DNA;        50 μg/ml tRNA; or 25 M sodium phosphate buffer pH 7.2; 1 mM        EDTA; 7% SDS

Hybridisation temperature:

-   -   T=65 to 68° C.    -   Wash buffer: 0.1×SSC; 0.1% SDS    -   Wash temperature: T=65 to 68° C.

In principle, nucleic acid molecules, which hybridise with the nucleicacid molecules according to the invention, can originate from any plantspecies, which codes an appropriate protein, preferably they originatefrom starch-storing plants, preferably from species of the (systematic)family Poacea, particularly preferably from Oryza sativa. Nucleic acidmolecules, which hybridise with the molecules according to theinvention, can, for example, be isolated from genomic or from cDNAlibraries. The identification and isolation of nucleic acid molecules ofthis type can be carried out using the nucleic acid molecules accordingto the invention or parts of these molecules or the reverse complementsof these molecules, e.g. by means of hybridisation according to standardmethods (see, for example, Sambrook et al., Molecular Cloning, ALaboratory Manual, 3rd edition (2001) Cold Spring Harbor LaboratoryPress, Cold Spring Harbor, N.Y. ISBN: 0879695773, Ausubel et al., ShortProtocols in Molecular Biology, John Wiley & Sons; 5th edition (2002),ISBN: 0471250929) or by amplification using PCR.

Nucleic acid molecules, which exactly or essentially have the nucleotidesequence specified under SEQ ID NO 1 or SEQ ID NO 3 or parts of thesesequences, can be used as hybridisation samples. The fragments used ashybridisation samples can also be synthetic fragments oroligonucleotides, which have been manufactured using establishedsynthesising techniques and the sequence of which correspondsessentially with that of a nucleic acid molecule according to theinvention. If genes have been identified and isolated, which hybridisewith the nucleic acid sequences according to the invention, then adetermination of this sequence and an analysis of the characteristics ofthe proteins coded by this sequence should be carried out in order toestablish whether an OK1 protein is involved. Homology comparisons onthe level of the nucleic acid or amino acid sequence and a determinationof the enzymatic activity are particularly suitable for this purpose.The activity of an OK1 protein can take place, for example, as describedabove under General Methods, Item 11. A preferred bonding affinity toP-starch in comparison with non-phosphorylated starch andautophosphorylation of an OK1 protein can be demonstrated using themethods already described above and under General Methods, Items 8 and12.

The molecules hybridising with the nucleic acid molecules according tothe invention particularly include fragments, derivatives and allelicvariants of the nucleic acid molecules according to the invention, whichcode an OK1 protein from plants, preferably from starch-storing plants,preferably from plant species of the genus Oryza, particularlypreferably from Oryza sativa or Arabidopsis thaliana. In conjunctionwith the present invention, the term “derivative” means that thesequences of these molecules differ at one or more positions from thesequences of the nucleic acid molecules described above and have a highdegree of identity with these sequences. Here, the deviation from thenucleic acid molecules described above can have come about, for example,due to deletion, addition, substitution, insertion or recombination.

In conjunction with the present invention, the term “identity” means asequence identity over the whole length of the coding region of at least60%, in particular an identity of at least 70%, preferably greater than80%, particularly preferably greater than 90% and especially of at least95%. In conjunction with the present invention, the term “identity” isto be understood to mean the number of amino acids/nucleotides(identity) corresponding with other proteins/nucleic acids, expressed asa percentage. Identity is preferably determined by comparing SEQ ID NO 2or SEQ ID NO 4 for amino acids or SEQ. ID NO 1 or SEQ ID NO 3 fornucleic acids with other proteins/nucleic acids with the help ofcomputer programs. If sequences that are compared with one another havedifferent lengths, the identity is to be determined in such a way thatthe number of amino acids, which have the shorter sequence in commonwith the longer sequence, determines the percentage quotient of theidentity. Preferably, identity is determined by means of the computerprogram ClustalW, which is well known and available to the public(Thompson et al., Nucleic Acids Research 22 (1994), 4673-4680). ClustalWis made publicly available by Julie Thompson(Thompson@EMBL-Heidelberg.DE) and Toby Gibson(Gibson@EMBL-Heidelberg.DE), European Molecular Biology Laboratory,Meyerhofstrasse 1, D 69117 Heidelberg, Germany. ClustalW can also bedownloaded from different Internet sites, including the IGBMC (Institutde Génétique et de Biologie Moléculaire et Cellulaire, B.P.163, 67404Illkirch Cedex, France; ftp://ftp-igbmc.u-strasbg.fr/pub/) and the EBI(ftp://ftp.ebi.ac.uk/pub/software/) as well as from all mirroredInternet sites of the EBI (European Bioinformatics Institute, WellcomeTrust Genome Campus, Hinxton, Cambridge CB10 1SD, UK).

Preferably, Version 1.8 of the ClustalW computer program is used todetermine the identity between proteins according to the invention andother proteins. In doing so, the following parameters must be set:KTUPLE=1, TOPDIAG=5, WINDOW=5, PAIRGAP=3, GAPOPEN=10, GAPEXTEND=0.05,GAPDIST=8, MAXDIV=40, MATRIX=GONNET, ENDGAPS(OFF), NOPGAP, NOHGAP.

Preferably, Version 1.8 of the ClustalW computer program is used todetermine the identity between the nucleotide sequence of the nucleicacid molecules according to the invention, for example, and thenucleotide sequence of other nucleic acid molecules. In doing so, thefollowing parameters must be set:

KTUPLE=2, TOPDIAGS=4, PAIRGAP=5, DNAMATRIX:IUB, GAPOPEN=10, GAPEXT=5,MAXDIV=40, TRANSITIONS: unweighted.

Furthermore, identity means that functional and/or structuralequivalence exists between the nucleic acid molecules concerned or theproteins coded by them. The nucleic acid molecules, which are homologousto the molecules described above and constitute derivatives of thesemolecules, are generally variations of these molecules, which constitutemodifications, which execute the same biological function. At the sametime, the variations can occur naturally, for example they can besequences from other plant species, or they can be mutants, whereinthese mutants may have occurred in a natural manner or have beenintroduced by objective mutagenesis. The variations can also besynthetically manufactured sequences. The allelic variants can be bothnaturally occurring variants and also synthetically manufacturedvariants or variants produced by recombinant DNA techniques. Nucleicacid molecules, which deviate from nucleic acid molecules according tothe invention due to degeneration of the genetic code, constitute aspecial form of derivatives.

The proteins coded from the different derivatives of nucleic acidmolecules according to the invention have certain commoncharacteristics. These can include, for example, biological activity,substrate specificity, molecular weight, immunological reactivity,conformation etc, as well as physical characteristics such as, forexample, the running behaviour in gel electrophoresis, chromatographicbehaviour, sedimentation coefficients, solubility, spectroscopiccharacteristics, stability; optimum pH, optimum temperature etc.Preferred characteristics of an OK1 protein have already been describedin detail above and are to be applied here accordingly.

The nucleic acid molecules according to the invention can be any nucleicacid molecules, in particular DNA or RNA molecules, for example cDNA,genomic DNA, mRNA etc. They can be naturally occurring molecules ormolecules manufactured by genetic or chemical synthesis methods. Theycan be single-stranded molecules, which either contain the coding or thenon-coding strand, or double-stranded molecules.

A further embodiment of the present invention relates to plant cellsaccording to the invention and plants according to the invention whereinthe foreign nucleic acid molecule is chosen from the group consisting of

-   a) T-DNA molecules, which, due, to integration into the plant    genome, lead to an increase in the expression of at least one OK1    gene (T-DNA activation tagging);-   b) DNA molecules that contain transposons, which lead to an increase    in the expression of an OK1 gene by way of integration into the    plant genome. (transposon activation tagging);-   c) DNA molecules that code an OK1 protein, and that are linked with    regulatory sequences, which provide the transcriptions in plant    cells, and which lead to an increase in the OK1 protein activity in    the cell.-   d) Nucleic acid molecules introduced by means of in vivo    mutagenesis, which lead to a mutation or an insertion of a    heterologous sequence in at least one endogenous gene coding an OK1    protein, wherein the mutation or insertion causes an increase in the    expression of a gene coding an OK1 protein.

In conjunction with the present invention, plant cells and plantsaccording to the invention can also be manufactured by the use ofso-called insertion mutagenesis (overview article: Thorneycroft et al.,2001, Journal of experimental Botany 52 (361), 1593-1601). Insertionmutagenesis is to be understood to mean particularly the insertion oftransposons or so-called transfer DNA (T-DNA) into a gene or near a genecoding for an OK1 protein, whereby, as a result of which, the activityof an OK1 protein in the cell concerned is increased.

The transposons can be both those that occur naturally in the cell(endogenous transposons) and also those that do not occur naturally insaid cell but are introduced into the cell (heterologous transposons) bymeans of genetic engineering methods, such as transformation of thecell, for example. Changing the expression of genes by means oftransposons is known to the person skilled in the art. An overview ofthe use of endogenous and heterologous transposons as tools in plantbiotechnology is presented in Ramachandran and Sundaresan (2001, PlantPhysiology and Biochemistry 39, 234-252).

T-DNA insertion mutagenesis is based on the fact that certain sections(T-DNA) of Ti plasmids from Agrobacterium can integrate into the genomeof plant cells. The place of integration in the plant chromosome is notdefined, but can take place at any point. If the T-DNA integrates into apart of the chromosome or near a part of the chromosome, whichconstitutes a gene function, then this can lead to an increase in thegene expression and thus also to a change in the activity of a proteincoded by the gene concerned.

Here, the sequences inserted into the genome (in particular transposonsor T-DNA) are distinguished by the fact that they contain sequences,which lead to an activation of regulatory sequences of an OK1 gene(“activation tagging”).

Plant cells and plants according to the invention can be produced bymeans of the so-called “activation tagging” method (see, for example,Walden et al., Plant J. (1991), 281-288; Walden et al., Plant Mol. Biol.26 (1994), 1521-1528). These methods are based on activating endogenouspromoters by means of “enhancer” sequences, such as the enhancer of the35S RNA promoter of the cauliflower mosaic virus, or the octopinesynthase enhancer.

In conjunction with the present invention, the term “T-DNA activationtagging” is to be understood to mean a T-DNA fragment, which contains“enhancer” sequences and which leads to an increase in the activity ofat least one OK1 protein by integration into the genome of a plant cell.

In conjunction with the present invention, the term “transposonactivation tagging” is to be understood to mean a transposon, whichcontains “enhancer” sequences and which leads to an increase in theactivity of at least one OK1 protein by integration into the genome of aplant cell.

In another embodiment, the DNA molecules according to the invention,which code an OK1 protein, are linked with regulatory sequences, whichinitiate transcription in plant cells (promoters) and lead to anincrease in OK1 protein activity in the cell. In this case, the nucleicacid molecules according to the invention are present in “sense”orientation to the regulatory sequences.

For expressing nucleic acid molecules according to the invention, whichcode an OK1 protein, these are preferably linked with regulatory DNAsequences, which guarantee transcription in plant cells. In particular,these include promoters. In general, any promoter that is active inplant cells is eligible for expression.

The promoter can be chosen so that expression takes place constitutivelyor only in a certain tissue, at a certain stage of the plant developmentor at a time determined by external influences. The promoter can behomologous or heterologous both with respect to the plant and withrespect to the nucleic acid molecule.

Suitable promoters are, for example, the promoter of the 35S RNA of thecauliflower mosaic virus and the ubiquitin promoter from maize forconstitutive expression, the patatin promoter B33 (Rocha-Sosa et al.,EMBO J. 8 (1989), 23-29) for tuber-specific expression in potatoes or apromoter, which only ensures expression in photosynthetically activetissues, e.g. the ST-LS1 promoter (Stockhaus et al., Proc. Natl. Acad.Sci. USA 84 (1987), 7943-7947; Stockhaus et al., EMBO J. 8 (1989),2445-2451) or, for endosperm-specific expression of the HMG promoterfrom wheat, the USP promoter, the phaseolin promoter, promoters of zeingenes from maize (Pedersen et al., Cell 29 (1982), 1015-1026; Quatroccioet al., Plant Mol. Biol. 15 (1990), 81-93), glutelin promoter (Leisy etal., Plant Mol. Biol. 14 (1990), 41-50; Zheng et al., Plant J. 4 (1993),357-366; Yoshihara et al., FEBS Lett. 383 (1996), 213-218) or shrunken-1promoter (Werr et al., EMBO J. 4 (1985), 1373-1380). However, promoterscan also be used, which are only activated at a time determined byexternal influences (see for example WO 9307279). Promoters ofheat-shock proteins, which allow simple induction, can be of particularinterest here. Furthermore, seed-specific promoters can be used, such asthe USP promoter from Vicia faba, which guarantees seed-specificexpression in Vicia faba and other plants (Fiedler et al., Plant Mol.Biol. 22 (1993), 669-679; Baumlein et al., Mol. Gen. Genet. 225 (1991),459-467).

Furthermore, a termination sequence (polyadenylation signal) can bepresent, which is used for adding a poly-A tail to the transcript. Afunction in the stabilisation of the transcripts is ascribed to thepoly-A tail. Elements of this type are described in the literature (cf.Gielen et al., EMBO J. 8 (1989), 23-29) and can be exchanged at will.

Intron sequences can also be present between the promoter and the codingregion. Such intron sequences can lead to stability of expression and toincreased expression in plants (Callis et al., 1987, Genes Devel. 1,1183-1200; Luehrsen, and Walbot, 1991, Mol. Gen. Genet. 225, 81-93;Rethmeier, et al., 1997; Plant Journal. 12(4):895-899; Rose andBeliakoff, 2000, Plant Physiol. 122 (2), 535-542; Vasil et al., 1989,Plant Physiol. 91, 1575-1579; XU et al., 2003, Science in China Series.C Vol. 46 No. 6, 561-569). Suitable intron sequences are, for example,the first intron of the sh1 gene from maize, the first intron of thepolyubiquitin gene 1 from maize, the first intron of the EPSPS gene fromrice or one of the two first introns of the PAT1 gene from Arabidopsis.

Furthermore, plant cells according to the invention and plants accordingto the invention can be manufactured by means of so-called “in situactivation”. In this case, the introduced genetic modification effects achange in the regulatory sequences of endogenous OK1 genes, which leadsto an increased expression of OK1 genes. Preferably, the activation ofan OK1 gene takes place by “in vivo” mutagenesis of a promoter or of“enhancer” sequences of an endogenous OK1 gene. In doing so, a promoteror an “enhancer” sequence, for example, can be changed in such a waythat the mutation produced leads to an increased expression of an OK1gene in plant cells according to the invention or plants according tothe invention in comparison with the expression of an OK1 gene in wildtype plant cells or wild type plants. The mutation in a promoter or an“enhancer” sequence can also lead to OK1 genes in plant cells accordingto the invention or plants according to the invention being expressed ata time at which they would not be expressed in wild type plant cells orwild type plants.

In conjunction with the present invention, the term “OK1 gene” isunderstood to mean a nucleic acid molecule (cDNA, DNA), which codes anOK1 protein, preferably an OK1 protein from starch-storing plants, morepreferably from Arabidopsis thaliana, and most preferably from rice.

During so-called “in vivo” mutagenesis, a hybrid RNA-DNA oligonucleotide(“chimeroplast”) is introduced into plant cells by way of transformation(Kipp, P. B. et al., Poster Session at the “5th International Congressof Plant Molecular Biology, Sep. 21-27, 1997, Singapore; R. A. Dixon andC. J. Arntzen, Meeting report on “Metabolic Engineering in TransgenicPlants”, Keystone Symposia, Copper Mountain, Colo., USA, TIBTECH 15,(1997), 441-447; international patent WO 9515972; Kren et al.,Hepatology 25, (1997), 1462-1468; Cole-Strauss et al., Science 273,(1996), 1386-1389; Beetham et al., 1999, PNAS 96, 8774-8778).

A part of the DNA components of the RNA-DNA oligonucleotide ishomologous to a nucleic acid sequence of an endogenous OK1 gene, but, incomparison with the nucleic acid sequence of an endogenous OK1 gene, ithas a mutation or contains a heterologous region, which is surrounded bythe homologous regions.

By way of base pairing of the homologous regions of the RNA-DNAoligonucleotide and of the endogenous nucleic acid molecule, followed byhomologous recombination, the mutation contained in the DNA component ofthe RNA-DNA oligonucleotide or heterologous region can be transferredinto the genome of a plant cell. This leads to an increase in theactivity of one or more OK1 proteins.

All these methods are based on the introduction of a foreign nucleicacid molecule into the genome of a plant cell or plant and are thereforebasically suitable for the manufacture of plant cells according to theinvention and plants according to the invention.

Surprisingly, it has been found that plant cells according to theinvention and plants according to the invention synthesise a modifiedstarch in comparison with starch of corresponding wild type plant cellsor wild type plants that have not been genetically modified.

The plant cells according to the invention and plants according to theinvention synthesise a modified starch, which in its physical-chemicalcharacteristics, in particular the starch phosphate content or thephosphate distribution, is changed in comparison with the synthesisedstarch in wild type plant cells or plants, so that this is better suitedfor special applications.

As no enzymes have previously been described, which exclusivelyphosphorylate P-starch, it has also previously not been possible toincrease the starch phosphate content of already phosphorylated starchin plants over a certain level. This is now possible through the use ofa protein according to the invention or a nucleic acid according to theinvention for the genetic modification of plants.

It was not possible to distribute phosphates in starch synthesised fromplants either, due to a lack of means available. Due to the provision ofproteins and nucleic acids according to the present invention, it is nowpossible to alter the phosphate ratio in native starches as well.

Therefore, the present invention also includes plant cells and plantsaccording to the invention, which synthesise a modified starch incomparison with corresponding wild type plant cells and wild type plantsthat have not been genetically modified.

In conjunction with the present invention, the term “modified starch”should be understood to mean that the starch exhibits changedphysical-chemical characteristics in comparison to unmodified starch,which is obtainable from corresponding wild type plant cells or wildtype plants.

In an additional embodiment of the present invention, plant cells orplants according to the invention synthesise a starch, which contains ahigh content of starch phosphate and/or an altered phosphatedistribution in comparison to starch that has been isolated fromcorresponding wild type plant cells and wild type plants.

In conjunction with the current invention, the term “phosphatedistribution” should be understood to mean the proportion of starchphosphate bonded to a glucose molecule in the C-2 position, C-3position, or C-6 position, with respect to the total starch phosphatecontent in the starch.

In an additional embodiment of the present invention, plant cells orplants according to the invention synthesise a starch, which exhibits analtered ratio of C-3 phosphate to C-6 phosphate in comparison to starchfrom wild type plants that have not been genetically modified. Preferredhere are starches, which have an increased proportion of starchphosphate bonded in the C-3 position compared with starch phosphatebonded in the C-6 position in comparison with starches from wild typeplant cells and wild type plants that have not been geneticallymodified.

In conjunction with the present invention, the term “ratio of C-3phosphate to C-6 phosphate” should be understood to mean the amount ofstarch phosphate, of which starch phosphate bonded to analpha-1,4-glucan in the C-3 position or C-6 position, respectively,contributes to the sum of the starch phosphate bonded to thealpha-1,4-glucan in the C-3 position and C-6 position (C-3 position+C-6position).

Different methods of determining the amount of starch phosphate aredescribed. Preferably, the method of determining the amount of starchphosphate described by Ritte et al. (2000, Starch/Stärke 52, 179-185)can be used. Particularly preferably, the determination of the amount ofstarch phosphate by means of 31P-NMR is carried out according to themethod described by Kasemusuwan and Jane (1996, Cereal Chemistry 73,702-707).

Furthermore, an object of the invention is genetically modified plants,which contain plant cells according to the invention. These types ofplants can be produced from plant cells according to the invention byregeneration.

In principle, the plants according to the invention can be plants of anyplant species, i.e. both monocotyledonous and dicotyledonous plants.Preferably they are useful plants, i.e. plants, which are cultivated bypeople for the purposes of food or for technical, in particularindustrial purposes.

In a further embodiment, the plant according to the invention is astarch-storing plant. In conjunction with the present invention, theterm “starch-storing plants” means all plants with plant parts, whichcontain a storage starch, such as, for example, maize, rice, wheat, rye,oats, barley, cassava, potato, sago, mung bean, pea or sorghum.

In conjunction with the present invention, the term “potato plant” or“potato” means the plant species of the genus Solanum, particularlytuber-producing species of the genus Solanum, and in particular Solanumtuberosum.

In conjunction with the present invention, the term “wheat plant” meansplant species of the genus Triticum or plants resulting from crosseswith plants of the genus Triticum, particularly plant species of thegenus Triticum or plants resulting from crosses with plants of the genusTriticum, which are used in agriculture for commercial purposes, andparticularly preferably Triticum aestivum.

In conjunction with the present invention, the term “maize plant” meansplant species of the genus Zea, particularly plant species of the genusZea, which are used in agriculture for commercial purposes, particularlypreferably Zea mais.

In an additional embodiment, the present invention relates tostarch-storing plants according to the invention of the (systematic)family Poaceae. These are preferably maize or wheat plants.

The present invention also relates to propagation material of plantsaccording to the invention containing a plant cell according to theinvention.

Here, the term “propagation material” includes those constituents of theplant that are suitable for producing offspring by vegetative or sexualmeans. Cuttings, callus cultures, rhizomes or tubers, for example, aresuitable for vegetative propagation. Other propagation materialincludes, for example, fruits, seeds, seedlings, protoplasts, cellcultures, etc. Preferably, the propagation material is tubers andparticularly preferably grains, which contain endosperms.

In a further embodiment, the present invention relates to harvestableplant parts of plants according to the invention such as fruits, storageroots, roots, blooms, buds, shoots or stems, preferably seeds, grains ortubers, wherein these harvestable parts contain plant cells according tothe invention.

Furthermore, the present invention also relates to a method for themanufacture of a genetically modified plant according to the invention,wherein

-   a) a plant cell is genetically modified, whereby the genetic    modification leads to an increase in the activity of an OK1 protein    in comparison with corresponding wild type plant cells that have not    been genetically modified;-   b) a plant is regenerated from plant cells from Step a); and-   c) if necessary, further plants are produced with the help of the    plants according to Step b).

The genetic modification introduced into the plant cell according toStep a) can basically be any type of genetic modification, which leadsto an increase in the activity of an OK1 protein.

The regeneration of the plants according to Step (b) can be carried outusing methods known to the person skilled in the art (e.g. described in“Plant Cell Culture Protocols”, 1999, edt. by R. D. Hall, Humana Press,ISBN 0-89603-549-2).

The production of further plants according to Step (c) of the methodaccording to the invention can be carried out, for example, byvegetative propagation (for example using cuttings, tubers or by meansof callus culture and regeneration of whole plants) or by sexualpropagation. Here, sexual propagation preferably takes place undercontrolled conditions, i.e. selected plants with particularcharacteristics are crossed and propagated with one another. In thiscase, the selection is preferably carried out in such a way that furtherplants, which are obtained in accordance with Step c), exhibit thegenetic modification, which was introduced in Step a).

In a further embodiment of the method according to the invention, thegenetic modification consists in the introduction of a foreign nucleicacid molecule according to the invention into the genome of the plantcell, wherein the presence or the expression of said foreign nucleicacid molecule leads to increased activity of an OK1 protein in the cell.

In a further embodiment of the method according to the invention, thegenetic modification consists in the introduction of a foreign nucleicacid molecule into the genome of the plant cell, wherein the foreignnucleic acid molecule codes an OK1 protein.

In a further embodiment, the method according to the invention is usedfor manufacturing a genetically modified plant according to theinvention for producing starch-storing plants.

In a further embodiment, the method according to the invention is usedfor producing maize or wheat plants according to the invention.

In a further embodiment of the method according to the invention, theforeign nucleic acid molecule is chosen from the group consisting of

-   a) Nucleic acid molecules, which code a protein with the amino acid    sequence specified under SEQ ID NO 2 or SEQ ID NO 4;-   b) Nucleic acid molecules, which code a protein that includes the    amino acid sequence, which is coded by insertion into plasmid    A.t.-OK1-pGE or insertion into plasmid pMI50;-   c) Nucleic acid molecules, which code a protein, the amino acid    sequence of which has an identity of at least 60% with the amino    acid sequence specified under SEQ ID NO 2 or SEQ ID NO 4;-   d) Nucleic acid molecules, which code a protein, the sequence of    which has an identity of at least 60% of the amino acid sequence    that is coded by insertion into plasmid A.t.-OK1-pGEM or insertion    into plasmid pMI50;-   e) Nucleic acid molecules, which include the nucleotide sequence    shown under SEQ ID NO 1 or SEQ ID NO 3 or a complimentary sequence;-   f) Nucleic acid molecules, which include the nucleotide sequence of    insertion contained in the plasmid A.t.-OK1-pGEM or plasmid pMI50;-   g) Nucleic acid molecules, the nucleic acid sequence of which has an    identity of at least 70% with the nucleic acid sequences described    under a), b), e), or f);-   h) Nucleic acid molecules, which hybridise with at least one strand    of the nucleic acid molecules described under a), b), e), or f)    under stringent conditions;-   i) Nucleic acid molecules, the nucleotide sequence of which deviates    from the sequence of the nucleic acid molecules identified under a),    b), e), or f) due to the degeneration of the genetic code, and-   j) Nucleic acid molecules, which represent fragments, allelic    variants and/or derivatives of the nucleic acid molecules identified    under a), b), c), d), e), f), g), h) or i).

In a further embodiment of the method according to the invention, theforeign nucleic acid molecule is chosen from the group consisting of

-   a) T-DNA molecules, which lead to an increase in the expression of    an OK1 gene through integration into the plant genome (T-DNA    activation tagging);-   b) DNA molecules, which contain transposons that lead to an increase    in the expression of an OK1 gene through integration into the plant    genome (transposon activation tagging);-   c) DNA molecules, which code an OK1 protein and are linked to    regulatory sequences that guarantee (initiate) the transcriptions in    plant cells, and which lead to an increase in the activity of an OK1    protein in the cell;-   d) Nucleic acid molecules introduced by way of in vivo mutagenesis,    which lead to a mutation or an insertion in a heterologous sequence    in at least one endogenous OK1 gene, wherein the mutation or    insertion causes an increase in the expression of an OK1 gene.

In a further embodiment, the present invention relates to a methodaccording to the invention, wherein the genetically modified plantsyntheses a modified starch in comparison with starch from wild typeplants that have not been genetically modified.

In a further embodiment of the method according to the invention, theplants according to the invention synthesise a modified starch, whichhas a higher starch phosphate content and/or a modified phosphatedistribution in comparison with starch isolated from corresponding wildtype plants.

In a further embodiment of the method according to the invention, theplants according to the invention synthesise a modified starch, whichhas a modified ratio of C-3 phosphate to C-6 phosphate in comparisonwith starch from wild type plants that have not been geneticallymodified. Particularly preferred here are starches, which have anincreased proportion of starch phosphate bonded in the C-3 positioncompared with starch phosphate bonded in the C-6 position in comparisonwith starches from wild type plants that have not been geneticallymodified.

The present invention also relates to the plants obtainable by themethod according to the invention.

Surprisingly, it has been found that starch isolated from plant cellsaccording to the invention and plants according to the invention, whichhave an increased activity of an OK1 protein, synthesise a modifiedstarch.

In particular, the increased quantities of starch phosphate in starchesaccording to the invention provide the starches with surprising andadvantageous properties. Starches according to the invention have anincreased proportion of loaded groups due to the increased proportion ofstarch phosphate, which considerably affect the functional properties.Starch that contains loaded functional groups is particularly usable inthe paper industry, where it is utilised for paper coating. Paper, whichis coated with loaded molecules that also exhibit good adhesiveproperties, is particularly suitable for absorbing pigments, such asdye, printing inks, etc., for example.

The present invention also relates to modified starches obtainable fromplant cells according to the invention or plants according to theinvention, from propagation material according to the invention or fromharvestable plant parts according to the invention.

In a further embodiment, the present invention relates to modifiedstarch according to the invention from starch-storing plants, preferablyfrom starch-storing plants of the (systematic) family Poaceae,particularly preferably from maize or wheat plants.

Furthermore the present invention relates to a method for themanufacture of a modified starch including the step of extracting thestarch from a plant cell according to the invention or from a plantaccording to the invention, from propagation material according to theinvention of such a plant and/or from harvestable plant parts accordingto the invention of such a plant, preferably from starch-storing partsaccording to the invention of such a plant. Preferably, such a methodalso includes the step of harvesting the cultivated plants or plantparts and/or the propagation material of these plants before theextraction of the starch and, further, particularly preferably the stepof cultivating plants according to the invention before harvesting.

Methods for extracting starches from plants or from starch-storing partsof plants are known to the person skilled in the art. Furthermore,methods for extracting starch from different starch-storing plants aredescribed, e.g. in Starch: Chemistry and Technology (Publisher:Whistler, BeMiller and Paschall (1994), 2nd Edition, Academic Press Inc.London Ltd; ISBN 0-12-746270-8; see e.g. Chapter XII, Page 412-468:Maize and Sorghum Starches: Manufacture; by Watson; Chapter XIII, Page469-479: Tapioca, Arrowroot and Sago Starches: Manufacture; byCorbishley and Miller; Chapter XIV, Page 479-490: Potato starch:Manufacture and Uses; by Mitch; Chapter XV, Page 491 to 506: Wheatstarch: Manufacture, Modification and Uses; by Knight and Oson; andChapter XVI, Page 507 to 528: Rice starch: Manufacture and Uses; byRohmer and Klem; Maize starch: Eckhoff et al., Cereal Chem. 73 (1996),54-57, the extraction of maize starch on an industrial scale isgenerally achieved by so-called “wet milling”.). Devices, which are incommon use in methods for extracting starch from plant material areseparators, decanters, hydrocyclones, spray dryers and fluid bed dryers.

In conjunction with the present invention, the term “starch-storingparts” is to be understood to mean such parts of a plant in which, incontrast to transitory leaf starch, starch is stored as a deposit forsurviving for longer periods. Preferred starch-storing plant parts are,for example, tubers, storage roots and grains, particularly preferredare grains containing an endosperm, especially particularly preferredare grains containing an endosperm of maize or wheat plants.

Modified starch obtainable by a method according to the invention formanufacturing modified starch is also the subject matter of the presentinvention.

In a further embodiment of the present invention, the modified starchaccording to the invention is native starch.

In conjunction with the present invention, the term “native starch”means that the starch is isolated from plants according to theinvention, harvestable plant plants according to the invention,starch-storing parts according to the invention or propagation materialof plants according to the invention by methods known to the personskilled in the art.

Furthermore, the use of plant cells according to the invention or plantsaccording to the invention for manufacturing a modified starch are thesubject matter of the present invention.

The person skilled in the art knows that the characteristics of starchcan be changed by thermal, chemical, enzymatic or mechanical derivation,for example. Derived starches are particularly suitable for differentapplications in the foodstuffs and/or non-foodstuffs sector. Thestarches according to the invention are better suited to be an initialsubstance for the manufacture of derived starches than for conventionalstarches, since they exhibit a higher proportion of reactive functionalgroups due to the higher starch phosphate content.

The present invention therefore also relates to the manufacture of aderived starch, wherein modified starch according to the invention isderived retrospectively.

In conjunction with the present invention, the term “derived starch” isto be understood to mean a modified starch according to the invention,the characteristics of which have been changed after isolation fromplant cells with the help of chemical, enzymatic, thermal or mechanicalmethods.

In a further embodiment of the present invention, the derived starchaccording to the invention is starch that has been treated with heatand/or acid.

In a further embodiment, the derived starches are starch ethers, inparticular starch alkyl ethers, O-allyl ethers, hydroxylalkyl ethers,O-carboxylmethyl ethers, nitrogen-containing starch ethers,phosphate-containing starch ethers or sulphur-containing starch ethers.

In a further embodiment, the derived starches are cross-linked starches.

In a further embodiment, the derived starches are starch graft polymers.

In a further embodiment, the derived starches are oxidised starches.

In a further embodiment, the derived starches are starch esters, inparticular starch esters, which have been introduced into the starchusing organic acids. Particularly preferably these are phosphate,nitrate, sulphate, xanthate, acetate or citrate starches.

The derived starches according to the invention are suitable fordifferent applications in the pharmaceutical industry and in thefoodstuffs and/or non-foodstuffs sector. Methods for manufacturingderived starches according to the invention are known to the personskilled in the art and are adequately described in the generalliterature. An overview on the manufacture of derived starches can befound, for example, in Orthoefer (in Corn, Chemistry and Technology,1987, eds. Watson and Ramstad, Chapter 16, 479-499).

Derived starch obtainable by the method according to the invention formanufacturing a derived starch is also the subject matter of the presentinvention.

Furthermore, the use of modified starches according to the invention formanufacturing derived starch is the subject matter of the presentinvention.

Starch-storing parts of plants are often processed into flours. Examplesof parts of plants from which flours are produced, for example, aretubers of potato plants and grains of cereal plants. For the manufactureof flours from cereal plants, the endosperm-containing grains of theseplants are ground and strained. Starch is a main constituent of theendosperm. In the case of other plants, which do not contain endosperm,and which contain other starch-storing parts instead such as tubers orroots, for example, flour is frequently produced by mincing, drying, andsubsequently grinding the storing organs concerned. The starch of theendosperm or contained within starch-storing parts of plants is afundamental part of the flour, which is produced from those plant parts,respectively. The characteristics of flours are therefore affected bythe starch present in the respective flour. Plant cells according to theinvention and plants according to the invention synthesise a modifiedstarch in comparison with wild type plant cells and wild type plantsthat have not been genetically modified. Flours produced from plantcells according to the invention, plants according to the invention,propagation material according to the invention, or harvestable partsaccording to the invention, therefore exhibit modified properties. Theproperties of flours can also be affected by mixing starch with floursor by mixing flours with different properties.

Therefore, an additional subject of the invention relates to flours,which contain a starch according to the invention.

A further subject of the present invention relates to flours, which areproduced from plant cells according to the invention, plants accordingto the invention, from starch-storing parts of plants according to theinvention, from propagation material according to the invention, or fromharvestable plant parts according to the invention. Preferredstarch-storing parts of plants according to the invention are tubers,storage roots, and grains containing an endosperm. Tubers preferablycome from potato plants, and grains preferably come from plants of the(systematic) family Poaceae, while grains particularly preferably comefrom maize or wheat plants.

In conjunction with the present invention, the term “flour” is to beunderstood to mean a powder obtained by grinding plant parts. Plantparts are possibly dried before grinding, and minced and/or strainedafter grinding.

Flours according to the invention are characterised in that they containstarch, which exhibits a modified phosphate content and/or a modifiedphosphate distribution particularly due to its increased water bindingcapacity. This is desirable in the processing of flours in thefoodstuffs industry for many applications, and in particular in themanufacture of baked goods, for example.

A further subject of the present invention is a method for themanufacture of flours, including the step of grinding plant cellsaccording to the invention, plants according to the invention, parts ofplants according to the invention, starch-storing parts of plantsaccording to the invention, propagation material according to theinvention, or harvestable material according to the invention.

Flours can be produced by grinding starch-storing parts of plantsaccording to the invention. Methods for the manufacture of flours areknown to the person skilled in the art. A method for the manufacture offlours preferably includes the step of harvesting the cultivated plantsor plant parts and/or the propagation material or the starch-storingparts of these plants before grinding, and particularly preferablyincludes the additional step of cultivating plants according to theinvention before harvesting.

In conjunction with the present invention, the term “parts of plants”should be understood to mean all parts of the plants that, asconstituents, constitute a complete plant in their entirety. Parts ofplants are scions, leaves, rhizomes, roots, knobs, tubers, pods, seeds,or grains.

In a further embodiment of the present invention, the method for the offlours includes processing plants according to the invention,starch-storing plants according to the invention, propagation materialaccording to the invention, or harvestable material according to theinvention before grinding.

In this case, processing can be heat treatment and/or drying, forexample. Heat treatment followed by a drying of the heat-treatedmaterial is used in the manufacture of flours from storage roots ortubers such as potato tubers, for example, before grinding. The mincingof plants according to the invention, starch-storing parts of plantsaccording to the invention, propagation material according to theinvention, or harvestable material according to the invention beforegrinding can also represent processing in the sense of the presentinvention. The removal of plant tissue before grinding, such as e.g.grain husks, also represents processing before grinding in the sense ofthe present invention.

In a further embodiment of the present invention, the method for themanufacture of flours includes processing the ground product aftergrinding.

In this case, the ground product can be strained after grinding, forexample, in order to produce various types of flours, for example.

A further subject of the present invention is the use of geneticallymodified plant cells according to the invention or plants according tothe invention for the manufacture of flours.

It is also an object of the present invention to provide means such asDNA molecules, for example, for the production of plant cells accordingto the invention and plants according to the invention, which synthesisea modified starch in comparison with modified wild type plant cells orwild type plants that have not been genetically modified.

The present invention therefore also relates to nucleic acid molecules,which code for a protein with the enzymatic activity of an OK1 protein,chosen from the group consisting of

-   a) Nucleic acid molecules, which code a protein with the amino acid    sequence specified under SEQ ID NO 2 or SEQ ID NO 4;-   b) Nucleic acid molecules, which code a protein that includes the    amino acid sequence, which is coded by insertion into the plasmid    A.t.-OK1-pGEM or insertion into the plasmid pMI50;-   c) Nucleic acid molecules, which code a protein, the sequence of    which has an identity of at least 60% with the amino acid sequence    specified under SEQ ID NO 2 or SEQ ID NO 4;-   d) Nucleic acid molecules, which code a protein, the sequence of    which has an identity of at least 60% of the amino acid sequence,    which is coded by insertion into the plasmid A.t.-OK1-pGEM or    insertion into the plasmid DSM pMI50;-   e) Nucleic acid molecules, which include the nucleotide sequence    specified under SEQ ID NO 1 or SEQ ID NO 3 or a complimentary    sequence;-   f) Nucleic acid molecules, which include the nucleotide sequence of    the insertion contained in the plasmid A.t.-OK1-pGEM or the plasmid    pMI50;-   g) Nucleic acid molecules, which have an identity of at least 70%    with the nucleic acid sequences described under a), b), e), or f);-   i) Nucleic acid molecules, which hybridise with at least one strand    of the nucleic acid molecules described under a), b), e), or f)    under stringent conditions;-   h) Nucleic acid molecules, the nucleotide sequence of which deviates    from the sequence of the nucleic acid molecules specified under a),    b), e), or f) due to degeneration of the genetic code; and-   j) Nucleic acid molecule, which represent fragments, allelic    variants, and/or derivatives of the nucleic acid molecules specified    under a), b), c), d), e), f), g), h), or i).

Basically, nucleic acid molecules according to the invention, canoriginate from any plant, preferably they originate from starch-storingplants, preferably from potato, barley, sorghum, barley, wheat, or ricepants, particularly preferably from Arabidopsis plants or rice plants,and more particularly preferably from Oryza sativa.

Furthermore, the present invention relates to nucleic acid molecules ofat least 21, preferably more than 50 and particularly preferably morethan 200 nucleotides length, which specifically hybridise with at leastone nucleic acid molecule according to the invention. Here, specificallyhybridise means that these molecules hybridise with nucleic acidmolecules, which code a protein according to the invention, but not withnucleic acid molecules, which code other proteins. In particular, theinvention relates to such nucleic acid molecules, which hybridise withtranscripts of nucleic acid molecules according to the invention and, asa result, can hinder their translation. Such nucleic acid molecules,which specifically hybridise with the nucleic acid molecules accordingto the invention, can, for example, be constituents of antisense, RNAior co-suppression constructs or ribozymes, or can be used as primers forPCR amplification.

Furthermore, the invention relates to recombinant nucleic acid moleculescontaining a nucleic acid molecule according to the invention.

In conjunction with the present invention, the term “recombinant nucleicacid molecule” is to be understood to mean a nucleic acid molecule,which contains additional sequences in addition to nucleic acidmolecules according to the invention, which do not naturally occur inthe combination in which they occur in recombinant nucleic acidsaccording to the invention. Here, the abovementioned additionalsequences can be any sequences, preferably they are regulatory sequences(promoters, termination signals, enhancers), particularly preferablythey are regulatory sequences that are active in plant tissue, andespecially particularly preferably they are regulatory sequences thatare active in plant tissue, in which storage starch is synthesised.Methods for the creation of recombinant nucleic acid molecules accordingto the invention are known to the person skilled in the art, and includegenetic methods such as bonding nucleic acid molecules by way ofligation, genetic recombination, or new synthesis of nucleic acidmolecules, for example (see e.g. Sambrok et al., Molecular Cloning, ALaboratory Manual, 3rd edition (2001) Cold Spring Harbour LaboratoryPress, Cold Spring Harbour, N.Y. ISBN: 0879695773, Ausubel et al., ShortProtocols in Molecular Biology, John Wiley & Sons; 5th edition (2002),ISBN: 0471250929).

A further embodiment of recombinant nucleic acid molecules of thepresent invention are vectors, in particular plasmids, cosmids, viruses,bacteriophages, and other customary vectors in gene technology, whichcontain the nucleic acid molecules according to the invention describedabove.

In a further embodiment, the nucleic acid molecules according to theinvention contained in the vectors are linked with regulatory sequences,which initiate expression in prokaryotic or eukaryotic cells. Here, theterm “expression” can mean both transcription and translation. Thenucleic acid molecules according to the invention can have an in “sense”orientation and/or an “antisense” orientation with respect to theregulatory sequences.

Regulatory sequences for expression in prokaryotic organisms, e.g. E.coli, and in eukaryotic organisms are sufficiently described inliterature, in particular such for expression in yeast are described,such as e.g. Saccharomyces cerevisiae. An overview of various systemsfor expression for proteins in various host organisms can be found, forexample, in Methods in Enzymology 153 (1987), 383-516 and in Bitter etal. (Methods in Enzymology 153 (1987), 516-544).

A further subject of the present invention is a host cell, particularlya prokaryotic or eukaryotic cell, which is genetically modified with anucleic acid molecule according to the invention and/or with a vectoraccording to the invention, as well as cells that originate from thesetypes of host cells, and which contain the genetic modificationaccording to the invention.

In a further embodiment, the invention relates to host cells,particularly prokaryotic or eukaryotic cells, which were transformedwith a nucleic acid molecule according to the invention or with a vectoraccording to the invention, as well as host cells, which originate fromthese types of host cells, and which contain the described nucleic acidmolecules according to the invention or vectors.

The host cells can be bacteria cells (e.g. E. coli, bacteria of thegenus Agrobacterium, particularly Agrobacterium tumefaciens orAgrobacterium rhizogenes) or fungal cells (e.g. yeast, particularly S.cerevisiae, Agaricus, in particular Agaricus bisporus, Aspergillus,Trichoderma), as well as plant or animal cells. Here, the term“transforms” means that the cells according to the invention aregenetically modified with a nucleic acid molecule according to theinvention, inasmuch as they contain at least one nucleic acid moleculeaccording to the invention in addition to their natural genome. This canoccur in the cell freely, possibly as a self-replicating molecule, or itcan be stably integrated into the genome of the host cell.

The host cells of microorganisms are preferable. Within the framework ofthe present patent application, this is understood to include allbacteria and all protists (e.g. fungi, particularly yeasts and algae),as they are defined in Schlegel “General Microbiology” (Georg ThiemePublishing House (1985), 1-2), for example.

Further host cells according to the invention are plant cells. Inprinciple, these can be plant cells from any plant species, i.e. bothmonocotyledonous and dicotyledonous plants. These are preferably plantcells from agricultural useful plants, i.e. from plants, which arecultivated by humans for nutritional, technical, or particularlyindustrial purposes. The invention relates preferably to plant cells andplants from starch-storing plants (maize, rice, wheat, rye, oat, barley,cassava, potato, sago, mung bean, pea or sorghum); in particular, plantcells from plants of the (systematic) family Poacea, particularlypreferably plant cells from maize or wheat plants.

Compositions containing a nucleic acid molecule according to theinvention, recombinant nucleic acid molecule according to the inventionor a vector according to the invention are also the subject matter ofthe present invention. Compositions containing a nucleic acid moleculeaccording to the invention, a recombinant nucleic acid moleculeaccording to the invention, or a vector according to the invention, anda host cell are preferred. Particularly preferably, the host cell is aplant cell, more particularly preferably a cell from maize or wheatplants.

A further aspect of Compositions according to the invention relates tocompositions, which can be used for producing host cells according tothe invention, preferably for producing plant cells according to theinvention. Preferably, this is a composition containing a nucleic acidmolecule according to the invention, a recombinant nucleic acid moleculeaccording to the invention, or a vector according to the invention, anda biolistic carrier, which is suitable for the introduction of a nucleicacid molecule according to the invention into a host cell. Preferredbiolistic carriers are particles of tungsten, gold or syntheticmaterials.

A further embodiment of compositions according to the invention relatesto compositions containing a nucleic acid molecule according to theinvention, a recombinant nucleic acid molecule according to theinvention, or a vector according to the invention, and a plant cell anda synthetic cultivation medium. Preferably, such compositions alsocontain polyethylene glycol (PEG) in addition to nucleic acid moleculesaccording to the invention, plant cells, and a synthetic cultivationmedium. In the case of these compositions, the recombinant nucleic acidmolecule according to the invention occurs outside of the plant cell,i.e. it is located outside of the cell interior of the plant cell, whichis enclosed by a cytoplasmic membrane.

Synthetic culture media, which are suitable for the cultivation and/ortransformation of plant cells, are known to the person skilled in theart, and are sufficiently described in literature, for example. Manydifferent synthetic cultivation media are also available for purchase inthe specialised trade (e.g. DUCHEFA Biochemie B.V., Belgium).

A further embodiment of compositions according to the invention relatesto compositions, which are used for the identification of nucleic acidsaccording to the invention. Preferably, such compositions containadditional nucleic acid molecules, in addition to a nucleic acidmolecule according to the invention, a recombinant nucleic acid moleculeaccording to the invention, or a vector according to the invention,particularly nucleic acid molecules of plant origination, which canoccur in the form of genomic DNA, mRNA, or as clones in so-called DNAlibraries. DNA libraries, which occur as cosmids, phagmids, plasmids,YACs or BACs are preferred. The DNA libraries can contain both genomicDNA and cDNA. The nucleic acid molecules according to the invention,recombinant nucleic acid molecules according to the invention, or avector according to the invention are used in these compositions,preferably as a hybridisation sample.

A further embodiment of the present invention relates to a protein,which exhibits starch-phosphorylating activity, and which requiresphosphorylated starch as a substrate. Preferably, this is a protein,which exhibits phosphorylated starch phosphorylating activity, and whichrequires phosphorylated starch as a substrate.

A further embodiment of the present invention relates to a proteinaccording to the invention, which requires phosphorylated starch as asubstrate, and transfers a residual phosphate of ATP to phosphorylatedstarch. Preferably, a protein according to the invention transfers theresidual beta-phosphate of ATP to phosphorylated starch. Particularlypreferably, a protein according to the invention transfers the residualbeta-phosphate of the ATP to phosphorylated starch and the residualgamma-phosphate of ATP to water, and therefore possesses the activity ofa [phosphorylated-alpha-1,4-glucan]-water-dikinase or a[phosphorylated-starch]-water-dikinase.

A further embodiment of the present invention relates to a proteinaccording to the invention, which accumulates as a phosphorylatedintermediate product when transferring residual phosphate tophosphorylated starch.

A further embodiment of the present invention relates to a proteinaccording to the invention, which exhibits increased bonding activity tophosphorylated starch in comparison to non-phosphorylated starch.

A further embodiment of the present invention relates to a proteinaccording to the invention, which introduces more additional phosphatemonoester bonds in the C-3 position in comparison to phosphate monoesterbonds in the C-6 position of the glucose molecules of a phosphorylatedstarch.

Preferably, at least 30%, more preferably at least 60%, particularlypreferably at least 90%, and most preferably at least 120% morephosphate monoester bonds in the C-3 position of the glucose moleculesof a phosphorylated starch are introduced in comparison with thephosphate monoester bonds in the C-6 position of the glucose moleculesof a phosphorylated starch.

A further subject of the present invention relates to a proteinaccording to the invention, which exhibits a molecular weight derivedfrom the amino acid sequence of 120 kDa to 145 kDa, preferably from 120kDa to 140 kDa, particularly preferably from 125 kDa to 140 kDa, andmost particularly preferably from 130 kDa to 135 kDa.

A further embodiment of the present invention relates to a proteinaccording to the invention, which exhibits a phosphohistidine domain.The phosphohistidine domain preferably contains two residual histidines.

A further subject of the present invention is proteins according to theinvention chosen from the group consisting of

-   a) Proteins, which include the amino sequence specified under SEQ ID    NO 2 or SEQ ID NO 4;-   b) Proteins, which are coded by the coding region of the DNA    inserted into the plasmid A.t.-OK1-pGEM or pMI50; or-   c) Proteins, which exhibit an identity of at least 60% with the    amino acid sequence of the proteins specified under a) or b).

In a further embodiment, the present invention relates to proteins withphosphorylated starch phosphorylating activity, wherein the codedprotein exhibits an identity of at least 70%, preferably at least 80%,particularly preferably at least 90%, and more particularly preferablyat least 95% with the amino acid sequence specified under SEQ ID NO 2 orSEQ ID NO 4, or with the amino acid sequence of an OK1 protein coded bythe insertion into plasmid A.t.-OK1-pGEM or plasmid pMI50.

A further embodiment of the present invention relates to a proteinaccording to the invention, characterised in that the amino acidsequence coding the protein exhibits a phosphohistidine domain.Preferably, the protein according to the invention exhibits aphosphohistidine domain, which has an identity of at least 50%,particularly at least 60%, preferably at least 70%, particularlypreferably at least 80%, and more particularly preferably at least 90%with the amino acid sequence specified in SEQ ID NO 5.

In a further embodiment, the present invention relates to a proteinaccording to the invention, wherein the protein originates from anArabidopsis or a rice plant.

A further embodiment of the present invention relates to a protein,which exhibits increased bonding activity to phosphorylated starch incomparison with non-phosphorylated starch, wherein the bonding activityto phosphorylated starch is increased by at least three times,preferably at least four times, particularly preferably at least fivetimes, and more particularly preferably at least six times, incomparison to the bonding activity of a non-phosphorylated starch.

In a further embodiment, the invention also relates to proteins, whichare coded by nucleic acid molecules according to the invention.

Description of Sequences

-   SEQ ID NO 1: Nucleic acid sequence comprising the coding region of    the A.t.-OK1 proteins from Arabidopsis thaliana. This sequence is    inserted in the A.t.-OK1-pGEM and OK1-pDEST17 vectors.-   SEQ ID NO 2: Amino acid sequence coding the A.t.-OK1 protein from    Arabidopsis thaliana. This sequence can be derived from the nucleic    acid sequence specified under SEQ ID NO 1.-   SEQ ID NO 3: Nucleic acid sequence comprising the coding region of    the O.s.-OK1 protein from Oryza sativa. This sequence is inserted in    the pMI50 vector.-   SEQ ID NO 4: Amino acid sequence coding the O.s.-OK1 protein from    Oryza sativa. This sequence can be derived from the nucleic acid    sequence specified under SEQ ID NO 3.-   SEQ ID NO 5: Peptide sequence coding the phosphohistidine domain of    the OK1 proteins from Arabidopsis thaliana and Oryza sativa.

DESCRIPTION OF FIGURES

FIG. 1: Denaturing acrylamide gel for identifying proteins fromArabidopsis thaliana, which preferably bond to non-phosphorylated starchin comparison with phosphorylated starch. A standard protein molecularweight marker is shown in trace “M”. Proteins obtained after incubatingcontrol preparation C from Example 1d) are shown in trace “-”. Proteinextracts of Arabidopsis thaliana, obtained after incubation withnon-phosphorylated starch, isolated from leaves of an Arabidopsisthaliana sex1-3 mutant (Preparation B, example 1d), are shown in trace“K”. Protein extracts of Arabidopsis thaliana, obtained after incubationwith starch, isolated from leaves of an Arabidopsis thaliana sex1-3mutant, which was phosphorylated retrospectively in vitro with an R1protein (Preparation A, Example 1d), are shown in trace “P”. Oncompletion of electrophoresis, the acrylamide gel was stained withCoomassie Blue.

FIG. 2: Demonstration of autophosphorylation of the OK1 protein. FIG. 2A) shows a denaturing (SDS) acrylamide gel on completion ofelectrophoresis stained with Coomassie Blue. FIG. 2 B) shows theautoradiography of a denaturing (SDS) acrylamide gel. The same amountsof the same samples were applied to each of the two gels. M: Standardprotein molecular weight marker; R1: Sample from reaction vessel 1according to Example 7 (after incubating an OK1 protein with ATP); R2:Sample from reaction vessel 2 according to Example 7 (after incubatingan OK1 protein with ATP the protein was heated to 95° C.); R3: Samplefrom reaction vessel 3 according to Example 7 (after incubating an OK1protein with ATP the protein was incubated in 0.5 M HCl); R4: Samplefrom reaction vessel 4 according to Example 7 (after incubating an OK1protein with ATP the protein was incubated in 0.5 M NaOH).

FIG. 3: Demonstration of the starch-phosphorylating activity of an OK1protein (see Example 6). OK1 protein was incubated withnon-phosphorylated starch isolated from leaves of an Arabidopsisthaliana sex1-3 mutant (Preparation A) and starch isolated from leavesof an Arabidopsis thaliana sex1-3 mutant, which was phosphorylatedretrospectively in vitro with an R1 protein (Preparation B). PreparationC is the same as Preparation B, except that this Preparation C wasincubated without OK1 protein. Two independent tests were carried outfor each preparation (A, B, C) (Test 1 and Test 2). The respectiveamounts are shown graphically, measured in cpm (counts per minute), on³³P labeled phosphate, which were introduced into non-phosphorylatedstarch (Preparation A) and phosphorylated starch (Preparation B) by theOK1 protein

FIG. 4: Comparison of the C-atom positions of glucose molecules of thestarch, which was phosphorylated from an R1 protein and an OK1 proteinrespectively (see Example 9). OK1 protein (Preparation A) was incubatedin the presence of ATP labeled with ³³P with starch isolated from leavesof an Arabidopsis thaliana sex1-3 mutant, which was phosphorylatedretrospectively in vitro with an R1 protein. R1 protein (Preparation B)was incubated in the presence of ATP labeled with ³³P with starchisolated from leaves of an Arabidopsis thaliana sex1-3 mutant. Oncompletion of incubation, a total hydrolysis of the starch was carriedout and the hydrolysis products were separated by means of HPAEchromatography. As standard, glucose-6-phosphate and glucose-3-phosphatewere added to the hydrolysis products before separation. The hydrolysisproducts separated by means of HPAE chromatography were collected inindividual fractions. The added glucose-6-phosphate eluted with fraction15 and the added glucose-3-phosphate with fraction 17. The fractionsobtained were subsequently investigated for the presence ofradioactively labeled phosphate. The amount of ³³P labeled phosphatemeasured in the individual fractions, measured in cpm (counts perminute), which was introduced into the hydrolysis products of thephosphorylated starch by the OK1 protein or the R1 protein, is showngraphically.

FIG. 5 Demonstration of the autophosphorylation of the OK1 protein. FIG.5 A) shows a Western blot. FIG. 5 B) shows the autoradiography of adenaturing (SDS) acrylamide gel. The same amounts of the same sampleswere applied to each of the two gels. The OK1 protein was incubatedeither with randomised radioactively labeled ATP or with ATPspecifically radioactively labeled in the gamma position. On completionof incubation, the proteins were either heated to 30° C. or 95° C., orincubated in 0.5 M NaOH or 0.5 M HCl respectively.

FIG. 6 Demonstration of the transfer of the beta-phosphate residue ofATP to starch in a reaction catalysed by an OK1 protein. Either ATPspecifically labeled with ³³P in the gamma position or randomised ³³PATP was used to phosphorylate starch, which had been phosphorylated invitro by means of an R1 protein and isolated from leaves of anArabidopsis thaliana sex1-3 mutant, by means of an OK1 protein. No OK1protein was added in any of the experiments designated as “control”.Each preparation was tested twice, independently from one another. Theresults of both tests are shown.

FIG. 7 Western Blot analysis of protein extracts from plants using anantibody against the OK1 protein from Arabidopsis thaliana. Proteinextracts from leaves of the following plants are shown: Ara Arabidosisthaliana; 51, 54, 55, 67, 72, 73, 79, 62, 63, 64, 65, 69, 66, 68 areindependent lines of the transformation 385JH; D wildtype Solanumtuberosum cv Désirée.

GENERAL METHODS

In the following, methods are described, which can be used for carryingout methods described in the invention. These methods constitutespecific embodiments of the present invention but do not restrict thepresent invention to these methods. The person skilled in the art knowsthat he can implement the invention in the same way by modifying themethods described and/or by replacing individual parts of the methods byalternative parts of the methods.

1. Manufacture of Protein Extracts from Plant Tissue

a) Manufacture of Protein Extracts from Plant Tissue

Leaf material is frozen in liquid nitrogen immediately after harvesting,and subsequently homogenised in the mortar under liquid nitrogen. Thereduced leaf material is mixed with ca. 3.5-times the volume (withrespect to the weight of the leaf material used) of cold (4° C.) bindingbuffer and broken down for 2×10 s with an Ultraturrax (maximum speed).After the first treatment with an Ultraturrax, the reduced leaf materialis cooled on ice before the second treatment is carried out. The treatedleaf material is then passed through a 100-μm nylon mesh and centrifugedfor 20 min (50 ml centrifuge vessel, 20.000×g, 4° C.).

b) Precipitation of the Proteins Contained in the Protein Extracts

The supernatant obtained following centrifugation according to Step a)is removed and its volume determined. To precipitate proteins, ammoniumsulphate is added continuously to the supernatant over a period of 30minutes while stirring on ice down to a final concentration of 75%(weight/volume). The supernatant is subsequently incubated for a furtherhour on ice while stirring. The proteins precipitated from thesupernatant are pellitised at 20.000×g and 4° C. for 10 min and thepellets are, subsequently absorbed in 5 ml of binding buffer, i.e. theproteins present in the pellet are dissolved.

c) Desalting of the Precipitated Proteins

The dissolved proteins are desalted by means of a PD10 column filledwith Sephadex G25 (Amersham Bioscience, Freiburg, Prod. No. columns:17-0851-01, Prod. No. Sephadex G25-M: 17-0033-01) at a temperature of 4°C., i.e. the ammonium sulphate used under Step b) for precipitation isalso separated from the dissolved protein. The PD10 column isequilibrated with binding buffer before the proteins dissolved inaccordance with Step b) are applied. For this purpose, 5 ml of bindingbuffer are spread over the column in each case. Subsequently, 2.5 ml ofthe protein solution obtained in accordance with Step b) are added toeach column before proteins are eluted from the column with 3.5 mlbinding buffer.

d) Determination of the Protein Concentration

The protein concentration is determined with a Bradford assay (Biorad,Munich, Prod. No. 500-0006 (Bradford, 1976, Anal. Biochem. 72,248-254)).

e) Composition of the Binding Buffer [

Binding buffer:  50 mM HEPES/NaOH (or KOH), pH 7.2   1 mM EDTA   2 mMDithioerythritol (DTE)   2 mM Benzamidine   2 mM ε-Aminocapronic acid0.5 mM PMSF 0.02% Triton X-100

2. Isolation of Leaf Starch

a) Isolation of Starch Granules from Plant Tissues

Leaf material is frozen immediately after harvesting in liquid nitrogen.The leaf material is homogenised in portions in the mortar under liquidnitrogen and absorbed into a total of ca. 2.5-times the volume(weight/volume) of starch buffer. In addition, this suspension is againhomogenised in the Waring blender for 20 s at maximum speed. Thehomogenate is passed through a nylon mesh (100 μm mesh width) andcentrifuged for 5 minutes at 1.000×g. The supernatant with the solubleproteins is discarded.

b) Cleaning the Starch Isolated from the Plant Tissues

After removing the green material lying on top of the starch by rinsingoff the green material with starch buffer, the pellet containing thestarch obtained from Step a) is absorbed in starch buffer andsuccessively passed through nylon meshes with different mesh widths (inthe order of 60 μm, 30 μm, 20 μm). The filtrate is centrifuged using a10 ml Percoll cushion (95% (v/v) Percoll (Pharmacia, Uppsala, Sweden),5% (v/v) 0.5M HEPES-KOH pH7.2) (Correx tube, 15 min, 2.000×g). Thesediment obtained after this centrifugation is re-suspended once instarch buffer and centrifuged again (5 min, 1.000×g).

c) Removal of the Proteins Bonded to the Starch

Following Step b), starch granules are obtained, which contain proteinsbonded to the starch. The proteins bonded to the surface of the starchgranules are removed by incubating four times with 0.5% SDS (sodiumlauryl sulphate) for 10-15 minutes in each case at room temperatureunder agitation. Each washing step is followed by a centrifugation (5min, 5.000×g), in order to separate the starch granules from therespective wash buffer.

d) Purification of Starch that has been Freed of Proteins

The starch obtained from Step c), which has been freed from the proteinsbonded to its surface, is subsequently removed by incubating four timeswith wash buffer for 10-15 minutes in each case at room temperatureunder agitation. Each washing step is followed by a centrifugation (5min, 5.000×g), in order to separate the starch granules from therespective wash buffer. These cleaning steps serve mainly to remove theSDS used in the incubations in Step c).

e) Determination of the Concentration of Isolated Starch

The amount of starch isolated in Step d) is determined photometrically.After suitable dilution, the optical density of the starch suspension ismeasured against a calibration curve at a wavelength of 600 nm. Thelinear range of the calibration curve is located between 0 and 0.3extinction units.

To produce the calibration curves, starch, for example isolated fromleaves of an Arabidopsis thaliana sex1-3 mutant, is dried under vacuum,weighed and absorbed in a defined volume of water. The suspension soobtained is diluted with water in several steps in a ratio of 1 to 1 ineach case until a suspension of ca. 5 μg starch per ml of water isobtained. The suspensions obtained by the individual dilution steps aremeasured in the photometer at a wavelength of 600 nm. The absorptionvalues obtained for each suspension are plotted against theconcentration of starch in the respective suspension. The calibrationcurve obtained should follow a linear mathematical function in the rangefrom 0 μg starch per ml of water to 0.3 μg starch per ml of water.

f) Storage of Isolated Starch

The starch can either be used directly without further storage forfurther tests, or stored in aliquots in 1.5 mL Eppendorf vessels at −20°C. Both the frozen starch and the non-stored, freshly isolated starchcan be used, if required, for the methods described in the presentinvention relating to in vitro phosphorylation and/or binding test, forexample.

g) Composition of Buffers Used

1× starch buffer:

-   -   20 mM HEPES-KOH, pH 8.0    -   0.5% Triton X-100

Wash buffer:

-   -   50 mM HEPES/KOH, pH 7.2

3. Recombinant Expression of an Identified Starch-PhosphorylatingProtein

a) Manufacture of a Bacterial Expression Vector Containing a cDNA, whichCodes a Starch-Phosphorylating Protein

The cDNA coding a starch-phosphorylating protein can be amplified, forexample, using mRNA or poly-A-plus-mRNA from plant tissues as a“template”, by means of a polymerase chain reaction (PCR). For thispurpose, a reverse transcriptase is first used for the manufacture of acDNA strand, which is complementary to an mRNA, which codes astarch-phosphorylating protein, before the cDNA strand concerned isamplified by means of DNA polymerase. So-called “kits” containingsubstances, enzymes and instructions for carrying out PCR reactions areavailable for purchase (e.g. SuperScript™ One-Step RT-PCR System,Invitrogen, Prod. No.: 10928-034). The amplified cDNA coding astarch-phosphorylating protein can subsequently be cloned in a bacterialexpression vector, e.g. pDEST™ (17 (Invitrogen). pDEST™17 contains theT7 promoter, which is used to initiate the transcription of theT7-RNA-polymerase. Furthermore, the expression vector pDEST™17 containsa Shine Dalgarno sequence in the 5′-direction of the T7 promoterfollowed by a start codon (ATG) and by a so-called His tag. This His tagconsists of six codons directly following one another, which each codethe amino acid histidine and are located in the reading frame of thesaid start codon. The cloning of a cDNA coding a starch-phosphorylatingprotein in pDEST™17 is carried out in such a way that a translationalfusion occurs between the codons for the start codon, the His tag andthe cDNA coding a starch-phosphorylating protein. As a result of this,following transcription initiated on the T7 promoter, and subsequenttranslation, a starch-phosphorylating protein is obtained, whichcontains additional amino acids containing the His tag on itsN-terminus.

However, other vectors, which are suitable for expression inmicroorganisms, can also be used for the expression of astarch-phosphorylating protein. Expression vectors and associatedexpression strains are known to the person skilled in the art and arealso available for purchase from the appropriate dealer in suitablecombinations.

b) Manufacture of Expression Clones in Escherichia coli

First of all, an appropriate transformation-competent E. coli strain,which chromosomally codes a T7-RNA polymerase, is transformed with theexpression plasmid manufactured under Step a), and subsequentlyincubated overnight at 30° C. on culture medium solidified with agar.Suitable expression strains are, for example, BL21 strains (InvitrogenProd. No.: C6010-03, which chromosomally code a T7-RNA polymerase underthe control of an IPTG-inducible promoter (lacZ).

Bacteria colonies resulting from the transformation can be investigatedusing methods known to the person skilled in the art to see whether theycontain the required expression plasmid containing a cDNA coding thestarch-phosphorylating protein. At the same time, expression clones areobtained.

c) Expression of a Starch-Phosphorylating Protein in Escherichia coli

First, a preparatory culture is prepared. To do this, an expressionclone obtained in accordance with Step b) is seeded in 30 ml TerrificBroth (TB medium) containing an antibiotic for selection on the presenceof the expression plasmid, and incubated overnight at 30° C. underagitation (250 rpm).

Next, a main culture is prepared for the expression of astarch-phosphorylating protein. To do this, in each case, 1 literErlenmeyer flasks, each containing 300 ml of TB medium, pre-heated to30° C., and an antibiotic for selection on the presence of theexpression plasmid are each seeded with 10 ml of an appropriatepreparatory culture and incubated at 30° C. under agitation (250 rpm)until an optical density (measured at a wavelength of 600 nm; OD₆₀₀) ofca. 0.8 is achieved.

If, for the expression of a starch-phosphorylating protein, anexpression plasmid is used, in which the expression of thestarch-phosphorylating protein is initiated by means of an induciblesystem (e.g. the expression vector pDEST™17 in BL21 E. coli strains,inducible by means of IPTG), then on reaching an OD₆₀₀ of ca. 0.8, theinductor concerned (e.g. IPTG) is added to the main culture. Afteradding the inductor, the main culture is incubated at 30° C. underagitation (250 rpm) until an OD₆₀₀ of ca. 1.8 is achieved. The mainculture is then cooled for 30 minutes on ice before the cells of themain culture are separated from the culture medium by centrifugation (10minutes at 4.000×g and 4° C.).

4. Purification of a Starch-Phosphorylating Protein

a) Breaking Down of Cells Expressing a Starch-Phosphorylating Protein

The cells obtained in Step c), General Methods, Item 3 are re-suspendedin lysis buffer. In doing so, ca. 4 ml lysis buffer are added to about 1g of cells. The re-suspended cells are then incubated for 30 minutes onice before they are broken down with the help of an ultrasonic probe(Baudelin Sonoplus UW 2070, Baudelin electronic, Berlin, settings: Cycle6, 70%, 1 minute) under continuous cooling by means of the ice. Caremust be taken here to ensure that the cell suspension is not heated toomuch during the ultrasonic treatment. The suspension obtained after theultrasonic treatment is centrifuged (12 minutes at 20.000×g, 4° C.) andthe supernatant obtained after centrifugation is filtered using a filterwith a pore size of 45 μm.

b) Purification of the Starch-Phosphorylating Protein

If the starch-phosphorylating protein expressed in E. coli cells is afusion protein with a His tag, then cleaning can take place with thehelp of nickel ions, to which the His tag bonds with greater affinity.To do this, 25 ml of the filtrate obtained in Step d) is mixed with 1 mlNi-agarose slurry (Qiagen, Prod. No.: 30210) and incubated for 1 hour onice. The mixture of Ni-agarose slurry and filtrate is subsequentlyspread over a polystyrene column (Pierce, Prod. No.: 29920). Theproduct, which runs through the column, is discarded. The column is nextwashed by adding 8 ml of lysis buffer, the product, which runs throughthe column, again being discarded. Elution of the starch-phosphorylatingprotein then takes place by fractionated addition to the column of 1 mlE1 buffer twice, followed by 1 ml E2 buffer once and subsequently 1 mlE3 buffer five times. The product, which runs through the column, whichis produced by adding the individual fraction of the appropriate elutionbuffer (E1, E2, E3 buffer) to the column, is collected in separatefractions. Aliquots of these fractions are subsequently analysed bymeans of denaturing SDS acrylamide gel electrophoresis followed byCoomassie Blue colouring. The fractions, which contain thestarch-phosphorylating protein in sufficient quantity and satisfactorypurity, are cleaned and concentrated with the help of pressurisedfiltration at 4° C. Pressurised filtration can be carried out, forexample, with the help of an Amicon cell (Amicon Ultrafiltration Cell,Model 8010, Prod. No.: 5121) using a Diaflo PM30 membrane (Millipore,Prod. No.: 13212) at 4° C. Other methods known to the person skilled inthe art can also be used for concentration however.

c) Composition of Buffers Used

Lysis buffer:  50 mM HEPES 300 mM NaCl  10 mM Imidazole pH 8.0 (adjustwith NaOH) 1 mg/ml Lysozyme (add immediately before using the buffer) ¼tablet per 10 ml, protease inhibitors completely EDTA free, (Rocheproduct No.: 1873580, add immediately before using the buffer)

Elution buffer E1:  50 mM HEPES 300 mM NaCl  50 mM Imidazole pH 8.0(adjust with NaOH) Elution buffer E2:  50 mM HEPES 300 mM NaCl  75 mMImidazole pH 8.0 (adjust with NaOH) Elution buffer E3:  50 mM HEPES 300mM NaCl 250 mM Imidazole pH 8.0 (adjust with NaOH)

5. Recombinant Expression of an R1 Protein

The recombinant expression of an R1 protein is described in theliterature (Ritte et al., 2002, PNAS 99, 7166-7171; Mikkelsen et al.,2004, Biochemical Journal 377, 525-532), but can also be carried out inaccordance with the methods relating to the recombinant expression of astarch-phosphorylating protein described above under General Methods,Item 3.

6. Purification of an R1 Protein

Purification of an R1 protein is described in the literature (Ritte etal., 2002, PNAS 99, 7166-7171; Mikkelsen et al., Mikkelsen et al., 2004,Biochemical Journal 377, 525-532), but can also be carried out inaccordance with the methods relating to the cleaning of astarch-phosphorylating protein described above under General Methods,Item 4 if an R1 fusion protein, which contains a His tag, is produced byexpression of R1 in E. coli cells.

7. In-Vitro Manufacture of Phosphorylated Starch on the Basis ofNon-Phosphorylated Starch

a) In Vitro Phosphorylation of Non-Phosphorylated Starch

Starch, which does not contain starch phosphate (e.g. isolated fromleaves of Arabidopsis thaliana sex1-3 mutants with the help of themethods described above under General Methods, Item 2), is mixed with R1buffer and with purified R1 protein (ca. 0.25 μg R1 protein per mgstarch) in order to produce a starch content of 25 mg per ml. Thisreaction preparation is incubated overnight (approx. 15 hours) at roomtemperature under agitation. R1 bonded to the starch present in thereaction preparation is removed on completion of the reaction by washingfour times with ca. 800 μl 0.5% SDS in each case. Subsequently, the SDSstill present in the in vitro phosphorylated starch is removed bywashing five times with 1 ml wash buffer in each case. All washing stepstake place at room temperature for 10 to 15 minutes under agitation.Each washing step is followed by a centrifugation (2 min, 10.000×g), inorder to separate the starch granules from the respective SDS buffer orwash buffer.

b) Composition of Buffers Used

R1 buffer:  50 mM HEPES/KOH, pH 7.5   1 mM EDTA   6 mM MgCl₂ 0.5 mM ATPWash buffer:  50 mM HEPES/KOH, pH 7.2

8. Bonding of Proteins to Phosphorylated Starch and Non-PhosphorylatedStarch

a) Isolation of P-Starch Protein Complexes or Non-Phosphorylated StarchProtein Complexes

Ca. 50 mg P-starch or ca. 50 mg non-phosphorylated starch respectivelyare re-suspended in separate preparations in ca. 800 μl protein extractin each case. The protein concentration of the protein extracts shouldbe ca. 4 mg to 5 mg per ml in each case. Incubation is carried out onthe P-starch or non-phosphorylated starch with protein extracts for 15minutes under agitation at 4° C. On completion of the incubation, thereaction preparations are centrifuged out using a Percoll cushion (4 ml)(15 minutes, 3500 rpm, 4° C.). Proteins, which are not bonded tophosphorylated starch or to P-starch, are located in the supernatantafter centrifugation, and they can be removed using a Pasteur pipette.The supernatant is discarded. The sedimented pellet containing P-starchand non-phosphorylated starch, including the proteins bonded to therespective starches (P-starch protein complexes or non-phosphorylatedstarch protein complexes respectively), obtained after centrifugation iswashed twice with 1 ml of wash buffer in each case (see above, GeneralMethods under Item 7b) by incubating for 3 minutes at 4° C. in each caseunder agitation. Every washing step is followed by a centrifugation (5minutes, 8000 rpm, 4° C. in a table centrifuge, Hettich EBA 12R) inorder to separate the P-starch or non-phosphorylated starch respectivelyfrom the wash buffer.

b) Dissolving the Proteins Bonded in the P-Starch Protein Complexes orNon-Phosphorylated Starch Protein Complexes Respectively

The P-starch protein complexes or non-phosphorylated starch proteincomplexes obtained according to Step a) are re-suspended in approx. 150μl SDS test buffer in each case, and incubated for 15 minutes underagitation at room temperature. The P-starch or non-phosphorylated starchrespectively is subsequently removed from the dissolved proteins bycentrifugation (1 minute, 13,000 rpm, room temperature, Eppendorf tablecentrifuge). The supernatant obtained after centrifugation iscentrifuged again in order to remove all residue of P-starch ornon-phosphorylated starch (1 minute, 13,000 rpm, room temperature,Eppendorf table centrifuge), and then it is removed. As a result,dissolved proteins, which bond to the P-starch or non-phosphorylatedstarch respectively, are obtained.

c) Composition of Buffers Used

SDS test buffer: 187.5 mM Tris/HCl pH 6.8    6% SDS   30% Glycerine~0.015% Bromphenol blue   60 mM Dithioerythritol (DTE, add fresh!)Percoll: Percoll is dialysed overnight against a solution consisting of[missing word?] and 25 mM HEPES/KOH, pH 7.0

9. Separation of Proteins that Bond to P-Starch and/orNon-Phosphorylated Starch

The dissolved proteins obtained in Step c) under General Methods, Item 8relating to the bonding of proteins to P-starch or non-phosphorylatedstarch respectively are incubated for 5 minutes at 95° C. in each caseand subsequently separated with the help of denaturing polyacrylamidegel electrophoresis. In doing so, an equal volume is applied to theacrylamide gel in each case for the dissolved proteins obtained bybonding to P-starch and for those obtained by bonding tonon-phosphorylated starch. The gel obtained on completion ofelectrophoresis is stained at least overnight with colloidal Comassie(Roth, Karlsruhe, Roti-Blue Rod. No.: A152.1), and subsequentlyde-stained in 30% methanol, 5% acetic acid or in 25% methanol.

10. Identification and Isolation of Proteins Bonding to P-Starch and/orNon-Phosphorylated Starch

a) Identification of Proteins with Increased Bonding Activity withRespect to P-Starch in Comparison with Non-Phosphorylated Starch

Proteins, which, after separation by means of acrylamide gelelectrophoresis and subsequent visualisation by colouration (see above,General Methods, Item 9), exhibit an increased signal after bonding toP-starch in comparison with a corresponding signal after bonding tonon-phosphorylated starch, have increased bonding activity with respectto P-starch in comparison with non-phosphorylated starch. By this means,it is possible to identify proteins, which have increased bondingactivity with respect to P-starch in comparison with non-phosphorylatedstarch. Proteins, which have increased bonding activity with respect toP-starch in comparison with non-phosphorylated starch, are excised fromthe acrylamide gel.

Identification of the amino acid sequence of proteins, which haveincreased bonding activity with respect to P-starch in comparison withnon-phosphorylated starch Proteins identified in accordance with Step a)are digested with trypsin and the peptides obtained are analysed bymeans of MALDI-TOF to determine the masses of the peptides obtained.Trypsin is a sequence-specific protease, i.e. trypsin only splitsproteins at a specified position when the proteins concerned containcertain amino acid sequences. Trypsin always splits peptide bonds whenthe amino acids arginine and lysine follow one another starting from theN-terminus. In this way, it is possible to theoretically determine allpeptides that would be produced following the trypsin digestion of anamino acid sequence. From the knowledge of the amino acids coding thetheoretically determined peptides, the masses of the peptides, which areobtained after theoretical trypsin digestion, can also be determined.Databases (e.g. NCBInr http://prospector.ucsf.edu/ucsfhtml4.0/msfit.htm;Swissprot http://cbrg.int.ethz.ch/Server/MassSearch.html), which containinformation concerning the masses of peptides after theoretical trypsindigestion, can therefore be compared with the real masses of peptides ofunknown proteins obtained with MALDI-TOF-MS. Amino acid sequences, whichhave the same peptide masses after theoretical and/or real trypsindigestion, are to be looked upon as being identical. The databasesconcerned contain both peptide masses of proteins, the function of whichhas already been shown, and also peptide masses of proteins, which up tonow only exist hypothetically by derivation from amino acid sequencesstarting from nucleic acid sequences obtained in sequencing projects.The actual existence and the function of such hypothetical proteins hastherefore seldom been shown and, if there is a function at all, thenthis is usually based only on predictions and not on an actualdemonstration of the function.

Bands containing proteins identified in accordance with Step a) areexcised from the acrylamide gel; the excised acrylamide piece is reducedand destained by incubating for approximately half an hour at 37° C. inca. 1 ml 60% 50 mM NH₄HCO₃, 40% acetonitrile. The decolourising solutionis subsequently removed and the remaining gel dried under vacuum (e.g.Speedvac). After drying, trypsin solution is added to digest theproteins contained in the gel piece concerned. Digestion takes placeovernight at 37° C. After digestion, a little acetonitrile is added(until the acrylamide gel is stained white) and the preparation is driedunder vacuum (e.g. Speedvac). When drying is complete, just enough 5%formic acid is added to cover the dried constituents and they areincubated for a few minutes at 37° C. The acetonitrile treatmentfollowed by drying is repeated once more. The dried constituents aresubsequently absorbed in 0.1% TFA (trifluoroacetic acid, 5 μl to 10 μl)and dripped onto a carrier in ca. 0.5 μl portions. Equal amounts ofmatrix (ε-cyano-4-hydroxy-cinnamic acid) are also applied to thecarrier. After crystallising out the matrix, the masses of peptides aredetermined by means of MALDI-TOF-MS-MS (e.g. Burker Reflex™ II, BrukerDaltonic, Bremen). With the masses obtained, databases are searched foramino acid sequences, which give the same masses after theoreticaltrypsin digestion. In this way, amino acid sequences can be identified,which code proteins, which preferably bond to phosphorylatedalpha-1,4-glucans and/or which need P-alpha-1,4-glucans as a substrate.

11. Method for Demonstrating the Starch-Phosphorylating Activity of aProtein

a) Incubation of Proteins with P-Starch and/or Non-Phosphorylated Starch

In order to demonstrate whether a protein has starch-phosphorylatingactivity, proteins to be investigated can be incubated with starch andradioactively labeled ATP. To do this, ca. 5 mg of P-starch or ca. 5 mgof non-phosphorylated starch are incubated with the protein to beinvestigated (0.01 μg to 5.0 μg per mg of starch used) in 500 μlphosphorylation buffer for 10 minutes to 30 minutes at room temperatureunder agitation. The reaction is subsequently stopped by the addition ofSDS up to a concentration of 2% (weight/volume). The starch granules inthe respective reaction mixture are centrifuged out (1 minute,13.000×g), and washed once with 900 μl of a 2% SDS solution and fourtimes each with 900 μl of a 2 mM ATP solution. Every washing step iscarried out for 15 minutes at room temperature under agitation. Aftereach washing step, the starch granules are separated from the respectivewash buffer by centrifugation (1 min, 13.000×g).

In addition, when carrying out an experiment to demonstratestarch-phosphorylating activity of a protein, further reactionpreparations, which do not contain protein or contain inactivatedprotein, but which are otherwise treated in the same way as the reactionpreparations described, should be processed as so-called controls.

b) Determination of the Amount of Phosphate Residues Incorporated in theP-Starch and/or Non-Phosphorylated Starch Due to Enzymatic Activity

The starch granules obtained in accordance with Step a) can beinvestigated for the presence of radioactively labeled phosphateresidues. To do this, the respective starch is re-suspended in 100 μl ofwater and mixed with 3 ml of scintillation cocktail in each case (e.g.Ready Safe™, BECKMANN Coulter) and subsequently analysed with the helpof a scintillation counter (e.g. LS 6500 Multi-Purpose ScintillationCounter, BECKMANN COULTER™).

c) Identification of Proteins, which Preferably Use P-Starch as aSubstrate

If a protein is incubated in separate preparations, once with P-starchand once with non-phosphorylated starch, in accordance with the methoddescribed under a), then, by comparing the values for the presence ofstarch phosphate obtained according to Step b), it can be determinedwhether the protein concerned has incorporated more phosphate inP-starch in comparison with non-phosphorylated starch. In this way,proteins can also be identified, which can introduce phosphate intoP-starch but not into non-phosphorylated starch. That means proteins canbe identified, which require already phosphorylated starch as asubstrate for an additional phosphorylation reaction.

d) Composition of Buffers Used

Phosphorylation buffer: 50 mM HEPES/KOH, pH 7.5 1 mM EDTA 6 mM MgCl₂0.01 to 0.5 mM ATP 0.2 to 2 μCi per ml randomised ³³P-ATP(alternatively, ATP, which contains a phosphate residue, which isspecifically labeled in the beta position, can also be used)

In conjunction with the present invention, the term “randomised ATP” isto be understood to mean ATP, which contains labeled phosphate residuesboth in the gamma position and in the beta position (Ritte et al. 2002,PNAS 99, 7166-7171). Randomised ATP is also described in the scientificliterature as beta/gamma ATP. A method for manufacturing randomised ATPis described in the following.

i) Manufacture of Randomised ATP

The method described here for manufacturing randomised ATP with the helpof enzyme-catalysed reactions is based on the following reactionmechanisms:

1st Reaction step:γ³³P-ATP+AMP+Myokinase→β³³P-ADP+ADP(Adenosine-P-P-³³P+Adenosine-P→Adenosine-P-P+Adenosine-P-³³P)

2nd Reaction step:³³P-ADP+ADP+2PEP+Pyruvate kinase→β³³P-ATP+ATP+2 Pyruvate(Adenosine-P-P+Adenosine-P-³³P+2PEP→Adenosine-P-P-P+Adenosine-P-³³P-P+2Pyruvate)

The reaction equilibriums lie on the product side but, in spite of this,this reaction produces a mixture consisting mainly of β³³P-ATP and someγ³³P-ATP.

ii) Performing the First Reaction Step

ATP (100 μCi, 3000 Ci per mmol), which contains a phosphate residuelabeled with ³³P in the gamma position (Hartmann Analytic, 10 μCi/μl),is incubated with 2 μl myokinase (AMP-phosphotransferase, from rabbitmuscle; SIGMA, Prod. No.: M3003 3.8 mg/ml, 1,626 units/mg) in 90 μlrandomising buffer for 1 hour at 37° C. The reaction is subsequentlystopped by incubating for 12 minutes at 95° C. before the reactionpreparation is cleaned up by means of centrifugal filtration using aMicrocon YM 10 filter (Amicon, Millipore Prod. No. 42407) at 14.000×gfor at least 10 minutes.

iii) Performing the Second Reaction Step

2 μl pyruvate kinase (see below for how to manufacture an appropriatesolution) and 3 μl 50 mM PEP (phosphoenolpyruvate) are added to thefiltrate obtained in Step ii). This reaction mixture is incubated for 45minutes at 30° C. before the reaction is stopped by incubating at 95° C.for 12 minutes. The reaction mixture is subsequently centrifuged (2minutes, 12,000 rpm in an Eppendorf table centrifuge). The supernatantcontaining randomised ATP obtained after centrifugation is removed,aliquoted and can be stored at −20° C.

Producing the Pyruvate Kinase Solution

15 μl pyruvate kinase (from rabbit muscle, Roche, Prod. No. 12815), 10mg/ml, 200 units/mg at 25° C.) are centrifuged out, the supernatant isdiscarded and the pellet is absorbed in 27 μl pyruvate kinase buffer.

Iv) Buffers Used

Pyruvate kinase buffer:  50 mM HEPES/KOH pH 7.5  1 mM EDTA Randomisingbuffer: 100 mM HEPES/KOH pH 7.5  1 mM EDTA 10% Glycerol  5 mM MgCl₂  5mM KCl  0.1 mM ATP  0.3 mM AMP

12. Demonstrating the Autophosphorylation of a Protein

In order to demonstrate whether a protein has auto-phosphorylatingactivity, proteins to be investigated can be incubated withradioactively labeled ATP. To do this, proteins to be investigated (50μg to 100 μg) are incubated in 220 μl phosphorylation buffer (see above,Item 12 d), General Methods) for 30 minutes to 90 minutes at roomtemperature under agitation. The reaction is subsequently stopped by theaddition of EDTA up to a final concentration of 0.11 M. Ca. 2 μg to 4 μgof protein are separated with the help of denaturing polyacrylamide gelelectrophoresis (7.5% acrylamide gel). The gel obtained afterpolyacrylamide gel electrophoresis is subjected to autoradiography.Proteins, which exhibit a signal in the autoradiography, carry aradioactive phosphate residue.

13. Identification of the C-Atom Positions of the Glucose Molecules ofan Alpha-1,4-Glucan, in which Residual Phosphates are Introduced Througha Starch-Phosphorylating Protein

Which C-atom positions of the glucose molecules of an alpha-1,4-glucanare phosphorylated by a protein can be demonstrated by hydrolysis of thephosphorylated glucan obtained by means of an appropriate protein invitro, subsequent separation of the glucose monomers obtained afterhydrolysis, followed by measurement of the phosphate incorporated by anappropriate protein in certain fractions of the glucose molecules.

a) Total Hydrolysis of the Alpha-1,4-Glucans

Water suspensions containing alpha-1,4-glucan are centrifuged, thesedimented pellet subsequently re-suspended in 0.7 M HCl (Baker, foranalysis) and incubated for 2 hours at 95° C. under agitation. Oncompletion of incubation, the samples are briefly cooled and centrifuged(e.g. 2 minutes 10.000×g). The supernatant obtained is transferred to anew reaction vessel and neutralised by the addition of 2 M NaOH (Baker,for analysis). If a pellet remains, it is re-suspended in 100 μl ofwater and the quantity of labeled phosphate present therein isdetermined as a control.

The neutralised supernatant is subsequently centrifuged over a 10-kDafilter. By measuring an aliquot of the filtrate obtained, the quantityof labeled phosphate in the filtrate is determined with the help of ascintillation counter, for example.

b) Fractionation of the Hydrolysis Products and Determination of thePhosphorylated C-Atom Positions

The neutralised filtrates of the hydrolysis products obtained by meansof Step a) can be separated (when using radioactively labeled ATP about3000 cpm) with the help of high-pressure anion exchange chromatography(HPAE), for example. The neutralised filtrate can be diluted with H₂O toobtain the volume required for HPAE. In addition, glucose-6-phosphate(ca. 0.15 mM) and glucose-3-phosphate (ca. 0.3 mM) are added to theappropriate filtrates in each case as an internal control. Separation bymeans of HPAE can be carried out, for example, with the help of a DionexDX 600 Bio Lc system using a CarboPac PA 100 column (with appropriatepre-column) and a pulsed amperometric detector (ED 50). In doing so,before injecting the sample, the column is first rinsed for 10 minuteswith 99% eluent C and 1% eluent D. A sample volume of 60 μl is theninjected. The elution of the sample takes place under the followingconditions:

Flow rate: 1 ml per minute

Gradient: linearly increasing from 0 minutes to 30 minutes

Eluent C Eluent D  0 minutes 99% 1% 30 minutes 0% 100% 35 minutes 0%100% Run terminated

The hydrolysis products eluted from the column are collected inindividual fractions of 1 ml each. As, in each case, non-labeledglucose-3-phosphate (Ritte et al. 2002, PNAS 99, 7166-7171) andnon-labeled glucose-6-phosphate (Sigma, Prod. No.: G7879) have beenadded to the injected samples of hydrolysis products as internalstandards, the fractions, which contain either glucose-3-phosphate orglucose-6-phosphate, can be determined by means of pulsed amperometricdetection. By measuring the amount of labeled phosphates in theindividual fractions and subsequently comparing with the fractions,which contain glucose-3-phosphate or glucose-6-phosphate, this can beused to determine those fractions, in which labeled glucose-6-phosphateor labeled glucose-3-phosphate is contained. The amount of labeledphosphate in the fraction concerned is determined. From the ratios ofthe amounts of glucose-3-phosphate to glucose-6-phosphate measured forlabeled phosphate in the individual hydrolysis products, it can now bedetermined which C-atom position is preferably phosphorylated by analpha-1,4-glucan phosphorylating enzyme.

c) Buffers Used:

Eluent C:

-   -   100 mM NaOH

Eluent D:

-   -   100 mM NaOH    -   500 mM sodium acetate

14. Transformation of Rice Plants

Rice plants were transformed according to the method described by Hieiet al. (1994, Plant Journal 6(2), 271-282).

15. Transformation of Potato Plants

Potato plants were transferred with the help of agrobacterium, asdescribed by Rocha-Sosa et al. (EMBO J. 8, (1989), 23-29).

16. Transformation of Wheat Plants

Wheat plants were transformed according to the method described byBecker et al. (1994, Plant Journal 5, 299-307).

17. Transformation of Maize Plants

Immature embryos of maize plants of line A188 were transformed accordingto the method described by Ishida et al. (1996, Nature Biotechnology 14,745-750).

18. Determination of Starch Phosphate Content

Determination of the C-6 Phosphate Content

In the starch, the C2, C3, and C6 positions of the glucose units can bephosphorylated. For determination of the C6-P content of the starch, 50mg of starch are hydrolysed in 500 μl 0.7 M HCl 4 h at 95° C.Subsequently, the preparations are centrifuged for 10 minutes at 15500g, and the supernatant is removed. 7 μl of supernatant is mixed with 193μl imidazole buffer (100 mM imidazole, pH 7.4; 5 mM MgCl₂, 1 mM EDTA,and 0.4 mM NAD). The measurement was taken in the photometer at 340 nm.After establishing a base absorption, the enzyme reaction is started byadding two units of glucose-6-phosphate dehydrogenase (of Leuconostocmesenteroides, Boehringer Mannheim). The change in absorption isdirectly proportional to the concentration of the G-6-P content in thestarch.

b) Determination of the Total Phosphate Content

The determination of the total phosphate content occurs according to theAmes method (Methods in Enzymology VIII, (1966), 115-118).

Approximately 50 mg starch is mixed with 30 μl of ethanolic magnesiumnitrate solution, and incinerated for three hours at 500° C. in themuffle oven. The residue is mixed with 300 μl 0.5 M hydrochloric acid,and incubated for 30 minutes at 60° C. Subsequently, an aliquot isfilled to 300 μl with 0.5 M hydrochloric acid, poured into a mixture of100 μl 10% ascorbic acid and 600 μl 0.42% ammonium molybdate in 2 Msulphuric acid, and incubated for 20 minutes at 45° C.

c) Determination of the Content of C-6 Phosphate and C-3 Phosphate

For the determination of the phosphate content, which is bonded in theC-6 position and the C-3 position of the glucose molecules of analpha-1,4-glucan, the respective glucans can be separated using totalhydrolysis according to the HPAE methods listed under General Methods13. The quantities of glucose-6-phosphate and glucose-3-phosphate can bedetermined through integration of the individual peak areas obtainedafter HPEA separation. By comparing the peak surfaces obtained forglucose-6-phosphate in unknown samples with peak surfaces that wereobtained after HPEA separation, having known quantities ofglucose-6-phosphate and glucose-3-phosphate, the quantity ofglucose-6-phosphate and glucose-3-phosphate can be determined in thesamples to be examined.

EXAMPLES 1. Isolation of a Protein from Arabidopsis thaliana, whichExhibits Increased Bonding Activity to P-Starch in Comparison toNon-Phosphorylated Starch

a) Manufacture of protein extracts from Arabidopsis thaliana

Protein extracts were produced from approximately 7 g of leaves (freshweight) of Arabidopsis thaliana (Okotyp Columbia, Col-O) according toGeneral Methods, Item 1.

b) Isolation of starch granules from leaves of sex1-3 mutants ofArabidopsis thaliana

Starch granules were isolated from about 20 g (fresh weight) of leavesof a sex1-3 mutant of Arabidopsis thaliana according to the methoddescribed under General Methods, Item 2.

c) In vitro phosphorylation of starch, isolated from a sex1-3 mutant ofArabidopsis thaliana with purified R1 protein

Approximately 30 mg of non-phosphorylated starch, isolated from a sex1-3mutant of Arabidopsis thaliana, was phosphorylated by way of an R1protein recombinantly expressed and purified in E. coli according to themethod described under General Methods, Item 7. For the expression ofthe R1 protein in E. coli and for subsequent purification, the methoddescribed by Ritte et al. (2002, PNAS 99, 7166-7171) was used.

d) Isolation of proteins, which bond to P-starch and/ornon-phosphorylated starch Protein extracts of Arabidopsis thaliana,obtained in accordance with Step a), were incubated and washed in aPreparation A with 50 mg of the in vitro phosphorylated starchmanufactured in accordance with Step c) using the method described underGeneral Methods, Item 8a.

In a second Preparation B, protein extracts of Arabidopsis thaliana,obtained in accordance with Step a), were incubated and washed with 50mg of the non-phosphorylated starch manufactured in accordance with Stepb) using the method described under General Methods, Item 8a.

Subsequently, the proteins bonded to the P-starch of Preparation A andto the non-phosphorylated starch of Preparation B were dissolved inaccordance with the method described under General Methods, Item 8b.

In a third Preparation C, 50 mg of the in vitro phosphorylated starchmanufactured in accordance with Step c) were incubated and washed usingthe method described under General Methods, Item 8a. Preparation Ccontained no protein extracts however.

e) Separation of the proteins obtained in accordance with Step d) bymeans of acrylamide gel electrophoresis

The proteins of Preparations A, B and C obtained in Step d) wereseparated by means of a 9% acrylamide gel under denaturing conditions(SDS) using the method described under General Methods, Item 9, andsubsequently stained with Coomassie Blue. The stained gel is shown inFIG. 1. It can be clearly seen that a protein, which has a molecularweight of ca. 130 kDa in denaturing acrylamide gel with regard to aprotein standard marker (Trace M), preferably bonds to phosphorylatedstarch (Trace P) in comparison with non-phosphorylated starch (K).

f) Identification of the protein, which preferably bonds to P-starch incomparison with non-phosphorylated starch

The band of the protein with a molecular weight of ca. 130 kDaidentified in Step e) was excised from the gel. The protein wassubsequently released from the acrylamide as described under GeneralMethods, Item 10b, digested with trypsin and the peptide masses obtainedwere determined by means of MALD-TOF-MS. The so-called “fingerprint”obtained by MALDI-TOF-MS was compared with fingerprints of theoreticallydigested amino acid molecules in databases (Mascot:http://www.matrixscience.com/search_form_select.html; ProFound:http://129.85.19.192/profound_bin/WebProFound.exe; PepSea:http://195.41.108.38/PepSeaIntro.html). As such a fingerprint is veryspecific to a protein, it was possible to identify an amino acidmolecule. With the help of the sequence of this amino acid molecule, itwas possible to isolate a nucleic acid sequence from Arabidopsisthaliana coding an OK1 protein. The protein identified with this, methodwas designated as A.t.-OK1. Analysis of the amino acid sequence of theOK1 protein from Arabidopsis thaliana showed that this deviated from thesequence that was present in the database (NP 198009, NCBI). The aminoacid sequence shown in SEQ ID No 2 codes the A.t.-OK1 protein. SEQ ID No2 contains deviations when compared with the sequence in the database(Acc.: NP 198009.1, NCBI). The amino acids 519 to 523 (WRLCE) and 762 to766 (VRARQ) contained in SEQ ID No 2 are not in the sequence, which ispresent in the database (ACC.: NP 198009.1). Compared with Version 2 ofthe database sequence (Acc.: NP 198009.2), the amino acid sequence shownin SEQ ID NO 2 also contains the additional amino acids 519 to 523(WRLCE).

2. Cloning a cDNA, which Codes the Identified OK1 Protein

The A.t.-OK1 cDNA was isolated with the help of reverse PCR using mRNAisolated from leaves of Arabidopsis thaliana. To do this, a cDNA Strandwas synthesised by means of reverse transcriptase (SuperScript™First-Strand Synthesis System for RT PCR, Invitrogen Prod. No.:11904-018), which was then amplified using DNA polymerase (Expand HighFidelity PCR Systems, Roche Prod. No.: 1732641). The amplified productobtained from this PCR reaction was cloned in the vector pGEM®-T(Invitrogen Prod. No.: A3600). The plasmid obtained is designatedA.t.-OK1-pGEM, the cDNA sequence coding the A.t.-OK1 protein wasdetermined and is shown under SEQ ID NO. 1.

The sequence shown under SEQ ID NO 1 is not the same as the sequence,which is contained in the database. This has already been discussed forthe amino acid sequence coding an A.t.-OK1 protein.

Conditions Used for the Amplification of the cDNA Coding the A.t.-OK1Protein

First Strand Synthesis:

The conditions and buffer specified by the manufacturer were used. Inaddition, the reaction preparation for the first strand synthesiscontained the following substances:

3 μg Total RNA 5 μM 3′-Primer (OK1rev1: 5′-GACTCAACCACATAACACACAAAGATC)0.83 μM dNTP Mix

The reaction preparation was incubated for 5 minutes at 75° C. andsubsequently cooled to room temperature.

The 1^(st) strand buffer, RNase inhibitor, and DTT were then added andincubated for 2 minutes at 42° C. before 1 μL Superscript RT DNApolymerase was added and the reaction preparation was incubated for 50minutes at 42° C.

Conditions for the Amplification of the First Strand by Means of PCR:

1 μL of the reaction preparation of the first strand synthesis 0.25 μM3′Primer (OK1rev2: 5′-TGGTAACGAGGCAAATGCAGA) 0.25 μM 5′Primer (OK1fwd2:5′-ATCTCTTATCACACCACCTCCAATG)

Reaction Conditions:

Step 1 95° C. 2 min Step 2 94° C. 20 sec Step 3 62° C. 30 sec (Temp. percycle-0.67° C.) (30 s), 68° C. ( Step 4 68° C. 4 minutes Step 5 94° C.20 sec Step 6 56° C. 30 sec Step 7 68° C. 4 minutes Step 8 68° C. 10minutes

The reaction was first carried out in accordance with Steps 1 to 4. 10repeats (cycles) were carried out between Step 4 and Step 2, thetemperature of Step 3 being reduced by 0.67° C. after each cycle. Thiswas subsequently followed by the reaction in accordance with theconditions specified in Steps 5 to 8. 25 repeats (cycles) were carriedout between Step 7 and Step 5, the time of Step 7 being increased by 5sec on each cycle. On completion of the reaction, the reaction wascooled to 4° C.

3. Creation of a Vector for Recombinant Expression of the cDNA of theOK1 Protein

Following amplification by means of PCR by using the plasmidA.t.-OK1-pGEM as a template using Gateway Technology (Invitrogen), thesequence coding the OK1 protein from Arabidopsis thaliana was nextcloned in the vector pDONOR™201 (Invitrogen Prod. No.: 11798-014).pDONOR™201. Subsequently, the coding region of the OK1 protein from thevector obtained was cloned by sequence-specific recombination in theexpression vector pDEST17™ (Invitrogen Prod. No.: 11803-014). Theexpression vector obtained was designated as A.t.-OK1-pDEST™17. Thecloning resulted in a translational fusion of the cDNA coding theA.t-OK1 protein with the nucleotides present in the expression vectorpDEST™17. The nucleotides originating from the vector pDEST™17, whichare translationally fused with the cDNA coding the A.t.-OK1 protein,code 21 amino acids. These 21 amino acids include, amongst others, thestart codon (ATG) and a so-called His tag (6 histidine residues directlyafter one another). After translation of these translationally fusedsequences, this results in an A.t.-OK1 protein, which has the additional21 amino acids coded by nucleotides originating from the vector at itsN-terminus. The recombinant A.t.-OK1 protein resulting from this vectortherefore contains 21 additional amino acids originating from the vectorpDEST™17 at its N-terminus.

4. Heterologous Expression of the OK1 Protein in E. Coli

The expression vector A.t.-OK1-pDEST™17 obtained in accordance withExample 3 was transformed in the E. coli strain BL21 Star™ (DE3)(Invitrogen, Prod. No. C6010-03). A description of this expressionsystem has already been given above (see General Methods, Item 3).Bacteria clones, containing the vector A.t.-OK1-pDEST™17, resulting fromthe transformation were next used to manufacture a preparatory culture,which was subsequently used for inoculating a main culture (see GeneralMethods, Item 3c). The preliminary culture and the main culture wereeach incubated at 30° C. under agitation (250 rpm). When the mainculture had reached an OD₆₀₀ of ca. 0.8, the expression of therecombinant A.t.-OK1 protein was induced by the addition of IPTG(isopropyl-beta-D-thiogalactopyranoside) until a final concentration of1 mM was achieved. After the addition of IPTG, the main culture wasincubated at 30° C. under agitation (250 rpm) until an OD₆₀₀ of ca. 1.8was achieved. The main culture was then cooled for 30 minutes on icebefore the cells of the main culture were separated from the culturemedium by centrifugation (10 minutes at 4.000×g and 4° C.).

5. Purification of the Recombinantly Expressed OK1 Protein

The purification and concentration of the A.t.-OK1 protein from cellsobtained in accordance with Example 4 was carried out using the methoddescribed under General Methods, Item 4.

6. Demonstration of Starch-Phosphorylating Activity of the OK1 Protein

The starch-phosphorylating activity of the A.t.-OK1 protein wasdemonstrated in accordance with the method described under GeneralMethods, Item 11. In doing so, 5 μg of cleaned A.t.-OK1 Proteinmanufactured in accordance with Example 5 was in each case incubated ina Preparation A with 5 mg of starch isolated from a sex1-3 mutant ofArabidopsis thaliana in accordance with Example 1b) and in a PreparationB with 5 mg of starch obtained by enzymatic phosphorylation inaccordance with Example 1c), in each case in 500 μl of phosphorylationbuffer containing 0.05 mM radioactively (³³P) labeled, randomised ATP(in total 1,130,00 cpm, ca. 0.55 μCi) for 30 minutes at room temperatureunder agitation. A Preparation C was used as a control, which was thesame as Preparation B, except that it contained no OK1 protein, but wasotherwise treated in the same way as Preparations A and B. Two tests,which were independent from one another, were carried out for allpreparations (A, B, C).

Using a scintillation counter, the starches from Preparations A, B, andC were investigated for the presence of radioactively labeled phosphate(see General Methods, Item 11b). The results are shown in Table 1 and inFIG. 3.

TABLE 1 Demonstration of starch-phosphorylating activity of the OK1protein Measured radioactivity [cpm] Trial 1 Trial 2 Preparation A(non-phosphorylated starch + OK1) 42 47 Preparation B (phosphorylatedstarch + OK1) 7921 8226 Preparation C (phosphorylated starch without 5653 protein)

From the results obtained, it can be seen that the OK1 protein does nottransfer phosphate groups from ATP to starch when non-phosphorylatedstarch is provided as a substrate, as the proportion of phosphate groupstransferred to non-phosphorylated starch by means of an OK1 protein,measured in cpm, does not exceed the proportion of radioactively labeledphosphate groups in Preparation C (control). If, on the other hand,P-starch is provided as a substrate, the proportion of radioactivephosphate groups, measured in cpm, which are transferred from ATP toP-starch, is significantly higher. From this, it can be seen that theOK1 protein requires P-starch as a substrate and that non-phosphorylatedstarch is not accepted as a substrate by the OK1 protein.

If the test described above is carried out with ATP specifically labeledin the gamma position with ³³P, then it is not possible to establish anincorporation of radioactively labeled phosphate in the starch. Fromthis, it can be seen that the beta phosphate residue of ATP istransferred from an OK1 protein to starch. The results of such a testare shown in FIG. 6.

7. Demonstration of Autophosphorylation

Autophosphorylation of the A.t.-OK1 protein was demonstrated by means ofthe methods described above (see General Methods, Item 12). Here, 50 μgof purified A.t.-OK1 protein were incubated with radioactively labeled,randomised ATP in 220 μl of phosphorylation buffer (see above, GeneralMethods, Item 12d) at room temperature for 60 minutes under agitation.Subsequently, 100 μl in each case were removed from the incubationpreparations and transferred to four fresh reaction vessels. In reactionvessel 1, the reaction was stopped by the addition of 40 μl 0.11 M EDTA.Reaction vessel 2 was incubated at 95° C. for 5 minutes. HCl was addedto reaction vessel 3 up to a final concentration of 0.5 M, and NaOH wasadded to reaction vessel 4 up to a final concentration of 0.5 M.Reaction vessels 3 and 4 were each incubated for 25 minutes at 30° C.Subsequently, 50 μl in each case were removed from reaction vessels 1,2, 3 and 4, mixed with SDS test buffer and separated by means of SDSacrylamide gel electrophoresis (7.5% acrylamide gel). For this purpose,samples from the reaction vessels were applied to each of two identicalacrylamide gels. One of the gels obtained on completion ofelectrophoresis was subjected to autoradiography, while the second gelwas stained with Coomassie Blue.

In the gel stained with Coomassie Blue (see FIG. 2A), it can be clearlyseen that treatment with 0.5 M NaOH leads to a degradation of OK1protein. The OK1 protein must therefore be described as unstablecompared with NaOH. Incubations at 30° C., 95° C. and with 0.5 M HClshow that the OK1 protein is relatively stable under the statedincubation conditions. This can be concluded from the fact that, underthese incubation conditions, in each case approximately the same amountsof OK1 protein can be demonstrated in the gel concerned after colouringwith Coomassie Blue.

In the autoradiography (see FIG. 2B), it can be seen by comparison withthe phosphorylated OK1 protein incubated at 30° C. that an incubation ofthe phosphorylated OK1 protein at 95° C. leads to a significantreduction in the phosphate, which has bonded to the OK1 protein. Thebond between the phosphate residue and an amino acid of the OK1 proteinmust therefore be described as heat-unstable. Furthermore, a slightreduction of the phosphate bonded to the OK1 protein can also be seenfor the incubation with 0.5 M HCl and 0.5 M NaOH in comparison withphosphorylated OK1 protein incubated at 30° C. If the fact is taken intoaccount that the quantity of OK1 protein in the autoradiography aftertreatment with 0.5 M NaOH is significantly less than in the samplestreated with heat and acid on account of the instability of the OK1protein compared with NaOH, then it can be concluded that the bondbetween the phosphate residue and an amino acid of the OK1 protein willbe relatively stable with respect to bases. As the sample treated withacid contains approximately the same amounts of protein as the sampleincubated at 30° C. and at 95° C., and yet has a significantly lowersignal in the autoradiography than the sample treated at 30° C., it mustbe assumed that acid incubation conditions also split the bond between aphosphate residue and an amino acid of the OK1 protein to a certainextent. An instability in the bond between a phosphate residue and anamino acid of the OK1 protein could therefore also be established in thetests carried out. At the same time, the instability with respect toacids is significantly less labeled than the instability with respect toheat.

Bonds between the amino acids histidine and phosphate are heat-unstable,acid-unstable but base-stable (Rosenberg, 1996, Protein Analysis andPurification, Birkhauser, Boston, 242-244). The results described aboveare therefore an indication that a phosphohistidine is produced by theautophosphorylation of an OK1 protein.

If recombinantly expressed OK1 protein, as described above, is incubatedwith ATP specifically labeled with ³³P in the gamma position, then noautophosphorylation can be detected. FIG. 5A shows the amount of proteinin the respective reaction preparation that can still be demonstrated bymeans of Western blot analysis after the appropriate incubation steps.FIG. 5B shows an autoradiography of protein from the individual reactionpreparations. It can be seen that, when ATP specifically labeled in thegamma position is used, no autophosphorylation of the OK1 protein takesplace, whereas, when randomised ATP is used, autophosphorylation can bedemonstrated. This means that when an OK1 protein is autophosphorylated,the phosphate residue of the beta position of the ATP is covalentlybonded to an amino acid of the OK1 protein.

8. Demonstration of the C-Atom Positions of the Glucose Molecules ofStarch Phosphorylated by an OK 1 Protein

a) Manufacture of Phosphorylated Starch

Phosphorylated starch was manufactured in accordance with GeneralMethods, Item 7. For this purpose, 5 mg non-phosphorylated starch,isolated from leaves of a sex1-3 mutant of Arabidopsis thaliana wasreacted with 25 μg purified A.t.-OK1 protein in a Preparation A, and 5mg in vitro phosphorylated starch, originally isolated from leaves of asex1-3 mutant of Arabidopsis thaliana, was reacted with 5 μg purified R1protein in a second Preparation B. In each case, the reaction occurredin 500 μl phosphorylation buffer, which contained ³³P labeled ATP ineach case (ca. 2.5×10⁶ cpm), by way of incubation at room temperaturefor 1 hour under agitation. In addition, a control preparation was used,which contained 5 mg of starch isolated from leaves of a sex1-3 mutantof Arabidopsis thaliana and the said phosphorylation buffer, but noprotein. The control preparation was treated in exactly the same way asPreparations A and B. The individual reactions were stopped by adding125 μl 10% SDS in each case and washed with 900 μl in each case, oncewith 2% SDS, five times with 2 mM ATP and twice with H₂O. Acentrifugation was carried out after each washing step (2 minutes in anEppendorf table centrifuge at 13,000 rpm in each case). The starchpellets obtained were re-suspended in 1 ml H₂O in each case, 100 μl ofeach preparation was mixed after adding 3 ml of scintillation cocktail(Ready Safe™, BECKMANN), and the preparations were subsequently measuredwith the aid of a scintillation counter (LS 6500 Multi-PurposeScintillation Counter, BECKMANN COULTER™).

The measurement provided the following results:

Control:  63 cpm/100 μL  630 cpm/1000 μl Preparation A (OK1): 1351cpm/100 μl 13512 cpm/1000 μl Preparation B (R1): 3853 cpm/100 μl 38526cpm/1000 μl

b) Total Hydrolysis of the P-Starch

The suspensions of Preparations A, B and C obtained in accordance withStep a) were centrifuged again (5 minutes in an Eppendorf tablecentrifuge at 13,000 rpm), the pellets obtained re-suspended in 90 μl0.7 M HCl (Baker, for analysis) and subsequently incubated for 2 hoursat 95° C. Preparations A, B and C were then centrifuged again (5 minutesin an Eppendorf table centrifuge at 13,000 rpm), and the supernatanttransferred to a new reaction vessel. Sedimented residues of thepreparations were re-suspended in 100 ml H₂O in each case and after theaddition of 3 ml of scintillation cocktail (Ready Safe™, BECKMANN) weremeasured with the help of a scintillation counter (LS 6500 Multi-PurposeScintillation Counter, BECKMANN COULTER™). Significant amounts ofradioactivity could not be demonstrated in any of the residues, whichmean that all the hydrolysis products labeled with radioactive phosphatewere located in the supernatant.

This was followed by neutralisation of the individual supernatantscontaining the hydrolysis products by the addition in each case of 30 μl2 M NaOH (the amount of NaOH required for neutralisation was tested outin advance on blind samples): The neutralised hydrolysis products wereplaced on a 10 kDa Microcon filter, which had previously been rinsedtwice with 200 μl H₂O in each case, and centrifuged for ca. 25 minutesat 12,000 rpm in an Eppendorf table centrifuge. 10 μl were taken fromthe filtrate obtained (ca. 120 μl in each case) and, after the additionof 3 ml of scintillation cocktail (Ready Safe™, BECKMANN), were measuredwith the help of a scintillation counter (LS 6500 Multi-PurposeScintillation Counter, BECKMANN COULTER™). The determination of theactivity present in the individual preparations gave the followingresults:

Preparation A  934 cpm/10 μl 11,208 cpm/120 μl  93 cpm/μl (OK1):Preparation B (R1): 2518 cpm/10 μl 30,216 cpm/120 μl 252 cpm/μl

c) Separation of the Hydrolysis Products

The hydrolysis products obtained in accordance with Step b) wereseparated by means of HPAE using a Dionex system under the conditionsstated above (see General Methods, Item 13c). The samples for separatingthe filtered supernatants of Preparations A and B obtained in accordancewith Step b) were composed as follows:

Preparation A (OK1): 43 μl of the supernatant of Preparation A obtainedin accordance with Step b) (equivalent to ca. 4,000 cpm), 32 μl H₂O, 2.5μl 2.5 mM glucose-6-phosphate and 2.5 μl 5 mM glucose-3-phosphate (ΣVolume=80 μl).

Preparation B (R1): 16 μl of the supernatant of Preparation B obtainedin accordance with Step b) (equivalent to ca. 4,000 cpm), 59 μl H₂O, 2.5μl 2.5 mM glucose-6-phosphate and 2.5 μl 5 mM glucose-3-phosphate (ΣVolume=80 μl).

In each case, 60 μl, containing ca. 3,000 cpm, of the appropriatesamples were injected for separation by means of HPAE. The HPAE wascarried out in accordance with the conditions specified under Item 23c.After passing through the HPAE column, the elution buffer was collectedin fractions, each of 1 ml. Collection of the fractions was begun 10minutes after injecting the sample. Based on the signal received fromthe PAD detector used, the elution of glucose-6-phosphate was assignedto fraction 15 and the elution of glucose-3-phosphate to fraction 17. Ineach case, 500 μl of the individual fractions were mixed with 3 ml ofscintillation cocktail (Ready Safe™, BECKMANN) and subsequently measuredwith the help of a scintillation counter (LS 6500 Multi-PurposeScintillation Counter, BECKMANN COULTER™). The following measurementswere obtained for the individual fractions:

TABLE 4 Measured amounts of radioactivity [cpm] in individual fractionsof hydrolysis products obtained by hydrolysis of starch phosphorylatedby means of an OK1 protein or R1 protein. Total cpm per FractionPreparation Preparation A (OK1) B (R1) Fr 13 8.7 3.3 Fr 14 13.1 32.2 Fr15 (G6P) 207.3 1952.8 Fr 16 399.8 112.3 Fr 17 (G3P) 1749.2 801.6 Fr 18196.7 17.3 Fr 19 6.7 18.9 Total 2581.5 2938.3 Deposit 3000.0 3000.0Recovery 86.0% 97.9%

The results are also shown graphically in FIG. 5.

After phosphorylation of starch catalysed by R1 protein, ca. 66% of theradioactively labeled phosphate, with respect to the total measuredradioactive phosphate in the analysed fractions, eluted afterhydrolysing the starch with the fraction, which containedglucose-6-phosphate as standard, and ca. 27% with the fraction, whichcontained glucose-3-phosphate as standard. After phosphorylation ofstarch catalysed by OK1 protein, ca. 67% of the radioactively labeledphosphate, with respect to the total measured radioactive phosphate inthe analysed fractions, eluted after hydrolysing the starch with thefraction, which contained glucose-3-phosphate as standard, and ca. 8%with the fraction, which contained glucose-6-phosphate as standard. Fromthis, it can be concluded that glucose molecules of the starch of R1proteins are preferably phosphorylated in the C-6 position, whereas fromOK1 proteins glucose molecules of the starch are preferablyphosphorylated in the C-3 position.

9. Identification of an OK1 Protein in Rice

Using the methods described under General Methods, Items 1 to 13, it wasalso possible to identify a protein from Oryza sativa (variety M202),which transfers a phosphate residue from ATP to P-starch. The proteinwas designated as O.s.-OK1. Non-phosphorylated starch is not used by theO.s.-OK1 protein as a substrate, i.e. the O.s.-OK1 protein also does notneed P-starch as a substrate. The nucleic acid sequence defining theidentified O.s.-OK1 protein is shown under SEQ ID NO 3 and the aminoacid sequence coding the O.s.-OK1 protein is shown under SEQ ID NO. 4.The amino acid sequence coding the O.s.-OK1 protein shown under SEQ IDNO 4 has an identity of 57% with the amino acid sequence coding theA.t.-OK1 protein shown under SEQ ID NO 2. The nucleic acid sequencecoding the O.s.-OK1 protein shown under SEQ ID NO 3 has an identity of61% with the nucleic acid sequence coding the A.t.-OK1 protein shownunder SEQ ID NO 1.

Manufacture of the Plasmid pMI50 Containing the Nucleic Acid SequenceCoding an OK1 Protein from Oryza sativa

The vector pMI50 contains a DNA fragment, which codes the complete OK1protein from rice of the variety M202.

The amplification of the DNA from rice was carried out in fivesub-steps.

The part of the open reading frame from position 11 to position 288 ofthe sequence specified under SEQ DIE NO 3 was amplified with the help ofreverse transcriptase and polymerase chain reaction using the syntheticoligonucleotides Os_ok1-R9 (GGAACCGATAATGCCTACATGCTC) and Os_ok1-F6(AAAACTCGAGGAGGATCAATGACGTCGCTGCGGCCCCTC) as a primer on RNA of immaturerice seeds. The amplified DNA fragment was cloned in the vector pCR2.1(Invitrogen catalogue number K2020-20). The plasmid obtained wasdesignated as pML123.

The part of the open reading frame from position 250 to position 949 ofthe sequence specified under SEQ DIE NO 3 was amplified with the help ofreverse transcriptase and polymerase chain reaction using the syntheticoligonucleotides Os_ok1-F4 (CCAGGTTAAGTTTGGTGAGCA) and Os_ok1-R6(CAAAGCACGATATCTGACCTGT) as a primer on RNA of immature rice seeds. Theamplified DNA fragment was cloned in the vector pCR2.1 (Invitrogencatalogue number K2020-20). The plasmid obtained was designated aspML120.

The part of the open reading frame from position 839 to position 1761 ofthe sequence specified under SEQ DIE NO 3 was amplified with the help ofreverse transcriptase and polymerase chain reaction using the syntheticoligonucleotides Os_ok1-F7 (TTGTTCGCGGGATATTGTCAGA) and Os_ok1-R7(GACAAGGGCATCAAGAGTAGTATC) as a primer on RNA of immature rice seeds.The amplified DNA fragment was cloned in the vector pCR2.1 (Invitrogencatalogue number K2020-20). The plasmid obtained was designated aspML121.

The part of the open reading frame from position 1571 to position 3241of the sequence specified under SEQ DIE NO 3 was amplified with the helpof reverse transcriptase and polymerase chain reaction using thesynthetic oligonucleotides Os_ok1-F8 (ATGATGCGCCTGATAATGCT) andOs_ok1-R4 (GGCAAACAGTATGAAGCACGA) as a primer on RNA of immature riceseeds. The amplified DNA fragment was cloned in the vector pCR2.1(Invitrogen catalogue number K2020-20). The plasmid obtained wasdesignated as pML119.

The part of the open reading frame from position 2777 to position 3621was amplified with the help of polymerase chain reaction using thesynthetic oligonucleotides Os_ok1-F3 (CATTTGGATCAATGGAGGATG) andOs_ok1-R2 (CTATGGCTGTGGCCTGCTTTGCA) as a primer on genomic DNA of rice.The amplified DNA fragment was cloned in the vector pCR2.1 (Invitrogencatalogue number K2020-20). The plasmid obtained was designated aspML122.

The cloning together of the sub-parts of the open reading frame of OK1was carried out as follows.

A 700 base pair along ApaI fragment of pML120, containing part of theopen reading frame of OK1, was cloned in the ApaI site of pML121. Theplasmid obtained was designated as pMI47.

A 960 base pair long fragment containing the areas of vectors frompML120 and pML123 coding for OK1 was amplified by means of polymerasechain reaction. In doing so, the primers Os_ok1-F4 (see above) andOs_ok1-R9 (see above), each in a concentration of 50 nm, and the primersOs_ok1-F6 and Os_ok1-R6, each in a concentration of 500 nm, were used.The amplified DNA fragment was cloned in the vector pCR2.1 (Invitrogencatalogue number K2020-20). The plasmid obtained was designated aspMI44.

An 845 base pair long fragment of pML122 was re-amplified forintroducing a XhoI site after the stop codon with the primers Os_ok1-F3(see above) and Os_ok1-R2Xho (AAAACTCGAGCTATGGCTGTGGCCTGCTTTGCA) andcloned in the vector pCR2.1 (Invitrogen catalogue number K2020-20). Theplasmid obtained was designated as t pMI45.

A 1671 base pair long fragment containing part of the open reading frameof OK1 was obtained from pML119 by digesting with the restrictionenzymes SpeI and PstI. The fragment was cloned in pBluescript II SK+(Genbank Acc.: X52328). The plasmid obtained was designated as pMI46.

A 1706 base pair long fragment containing part of the open reading frameof OK1 was excised with the restriction enzymes SpeI and XhoI from pMI46and cloned in the vector pMI45, which had been excised with the samerestriction enzymes. The plasmid obtained was designated as pMI47.

A 146 base pair long fragment containing part of the open reading frameof OK1 was excised with the restriction enzymes AfIII/NotI from pMI43and cloned in the vector pMI44, which had been excised with the samerestriction enzymes. The plasmid obtained was designated as pMI49.

A 1657 base pair long fragment containing part of the open reading frameof OK1 was excised with the restriction enzymes NotI and NarI from thevector pMI49 and cloned in the vector pMI47, which had been excised withthe same restriction enzymes. The plasmid obtained was designated aspMI50 and contains the whole coding region of the OK1 protein identifiedin rice.

10. Production of an Antibody, which Specifically Recognises an OK1Protein

As an antigen, ca. 100 μg of purified A.t.-OK1 protein was separated bymeans of SDS gel electrophoresis, the protein bands containing theA.t.-OK1 protein were excised and sent to the company EUROGENTEC S.A.(Belgium), which carried out the manufacture of the antibody undercontract. Next, the preimmune serums of rabbits were investigated to seewhether they would already detect a protein from an A. t. total extractbefore immunisation with recombinant OK1. The preimmune serums of tworabbits detected no proteins in the range 100-150 kDa and were thuschosen for immunisation. 4 injections of 100 μg of protein (Tag 0, 14,28, 56) were given to each rabbit. 4 blood samples were taken from eachrabbit: (Tag 38, Tag 66, Tag 87 and the final bleeding). Serum, obtainedafter the first bleeding, already showed a specific reaction with OK1antigen in Western blot. However, in all further tests, the lastbleeding of a rabbit was used.

11. Production of Transgenic Rice Plants, which Exhibit IncreasedActivity of an OK1 Protein

a) Manufacture of the Plasmid pGlo-A.t.-OK1

The plasmid pIR94 was obtained by amplifying the promoter of theglobulin gene from rice by means of a polymerase chain reaction (30×20sec 94° C., 20 sec 62° C., 1 min 68° C., 4 mM Mg₂SO₄) with the primersglb1-F2 (AAAACAATTGGCGCCTGGAGGGAGGAGA) and glb1-R1(AAAACAATTGATGATCAATCAGACAATCACTAGAA) on the genomic DNA of rice of thevariety M202 with High Fidelity Taq Polymerase (Invitrogen, cataloguenumber 11304-011) and cloned in pCR2.1 (Invitrogen catalogue numberK2020-20).

The plasmid pIR115 was obtained by cloning a synthetic piece of DNAconsisting of the two oligonucleotides X1(TGCAGGCTGCAGAGCTCCTAGGCTCGAGTTAACACTAGTAAGCTTAATTAAGAT ATCATTTAC) andX2 (AATTGTAAATGATATCTTAATTAAGCTTACTAGTGTTAACTCGAGCCTAGGAGCTCTGCAGCCTGCA) in the vector pGSV71 excised with SdaI and MunI.

The plasmid pIR115 obtained was excised with SdaI, the protruding3′-ends smoothed with T4 DNA polymerase, and a HindIII/SphI fragmentfrom pBinAR (Höfgen and Willmitzer, 1990, Plant Science 66, 221-230)with a size of 197 base pairs, smoothed by means of T4 DNA polymeraseand containing the termination signal of the octopine synthase gene fromAgrobacterium tumefaciens, was inserted. The plasmid obtained wasdesignated as pIR96.

The plasmid pIR103 was obtained by cloning a 986 base pair long DNAfragment from pIR94 containing the promoter of the globulin gene fromrice, which was cloned in the plasmid pIR96.

pGSV71 is a derivative of the plasmid pGSV7, which is derived from theintermediate vector pGSV1. pGSV1 constitutes a derivative of pGSC1700,the construction of which has been described by Cornelissen andVanderwiele (Nucleic Acid Research 17, (1989), 19-25). pGSV1 wasobtained from pGSC1700 by deletion of the carbenicillin resistance geneand deletion of the T-DNA sequences of the TL-DNA region of the plasmidpTiB6S3.

pGSV7 contains the replication origin of the plasmid pBR322 (Bolivar etal., Gene 2, (1977), 95-113) as well as the replication origin of thePseudomonas plasmid pVS1 (Itoh et al., Plasmid 11, (1984), 206). pGSV7also contains the selectable marker gene aadA, from the transposonTn1331 from Klebsiella pneumoniae, which gives resistance against theantibiotics spectinomycin and streptomycin (Tolmasky, Plasmid 24 (3),(1990), 218-226; Tolmasky and Crosa, Plasmid 29(1), (1993), 31-40).

The plasmid pGSV71 was obtained by cloning a chimeric bar gene betweenthe border regions of pGSV7. The chimeric bar gene contains the promotersequence of the cauliflower mosaic virus for initiating thetranscription (Odell et al., Nature 313, (1985), 180), the bar gene fromStreptomyces hygroscopicus (Thompson et al., Embo J. 6, (1987),2519-2523) and the 3′-untranslated area of the nopaline synthase gene ofthe T-DNA of pTiT37 for terminating the transcription andpolyadenylation. The bar gene provides tolerance against the herbicideglufosinate ammonium.

A DNA fragment, which contains the sequence of the entire open readingframe of the OK1 protein from Arabidopsis, was excised from the vectorA.t.-OK1-pGEM, and cloned into the vector pIR103. For this purpose, theplasmid A.t.-OK1-pGEM was excised with the restriction enzyme Bsp1201,the ends were smoothed with T4-DNA polymerase, and re-excised with SalI.The DNA fragment coding the OK1 protein from Arabidopsis thaliana wascloned into the vector pIR103, which was excised with EcI136II and XhoI.The plasmid obtained was designated as

b) Transformation of Rice Plants

Rice plants (variety M202) were transformed with Agrobacterium(containing the plasmid pGlo-A.t.-OK1), using the method described byHiei et al. (1994, Plant Journal 6(2), 271-282).

c) Analysis of the Transgenic Rice Plants and the Starch Synthesisedfrom these

By way of quantitative RT PCR analysis, it was possible to identifyplants, which exhibit an expression of mRNA coding A.t.-OK1 protein.

Plants, which exhibit a detectable amount of mRNA coding A.t.-OK1protein in comparison to corresponding wild type plants, were grown inthe greenhouse. Grains of these plants were harvested. Starch, isolatedfrom these mature grains, showed an increased content of phosphatecovalently bonded to the respective starch in comparison to starch,which was isolated from grains of corresponding wild type plants.

12. Production of Transgenic Potato Plants, which Exhibit IncreasedActivity of an OK1 Protein

a) Manufacture of the Plasmid pBinB33-Hyg

Starting with the plasmid pBinB33, the EcoRI-HindIII fragment containingthe B33 promoter, a part of the polylinker, and the ocs-terminator wereexcised and spliced into the correspondingly excised vector pBIB-Hyg(Becker, 1990, Nucl. Acids Res. 18, 203). The plasmid pBinB33 wasobtained by splicing the promoter of the patatin gene B33 from Solanumtuberosum (Rocha-Sosa et al., 1989) as a DraI fragment(nucleotide-1512-+14) into the vector pUC19 excised with SstI, the endsof which had been smoothed with the help of the T4 DNA polymerase. Thisresulted in the plasmid pUC19-B33. The B33 promoter was excised fromthis plasmid with EcoRI and SmaI and spliced into the correspondinglyexcised vector pBinAR (Höfgen and Willmitzer, 1990, Plant Science 66,221-230). This resulted in the plant expression vector pBinB33.

b) Manufacture of the Vector A.t.-OK1-pBinB33-Hyg

The coding sequence of the A.t.-OK1 protein was excised with therestriction endonucleases Bsp120I and SaII from the plasmidA.t.-OK1-pGEM and spliced into the vector pBinB33-Hyg excised with SmaIand SaII. The plasmid obtained was designated as A.t.-OK1-pBinB33-Hyg.

c) Transformation of Potato Plants

Agrobacterium tumefaciens (strain GV2260) was transformed with theplasmid OK1-pBinB33-Hyg. Subsequently, potato plants of the varietyDésirée were transformed with the help of the Agrobacterium tumefacienscontaining the plasma A.t.-OK1-pBinB33-Hyg in accordance with the methoddescribed by Rocha-Sosa et al. (EMBO J. 8, (1989), 23-29), and theplants were regenerated. The plants obtained from this transformationevent were designated 385JH.

d) Analysis of the Transgenic Potato Plants and the Starch Synthesisedfrom these

By means of Northern Blot analysis, it was possible to identify plantsof the transformation event 385JH, which exhibit an expression of mRNA,coding the A.t-OK1 protein.

A Western Blot analysis, which was performed with the antibody describedunder Example 10, confirmed, that plants of the transformation event385JH, in which mRNA of the heterologously expressed OK1 protein wasdetected, also exhibit an increased quantity of OK1 protein incomparison to wild type plants that have not been transformed. FIG. 7exemplary shows the detection of the A.t.-OK1 Protein in single plantsfrom the transformation event 385JH by means of Western Blot analysis.For induction of the B33 Promotor in leaf tissue single lines of thetransformation event 385JH were cultivated on solidified Musharige Skoogmedium containing 100 mM sucrose in tissue culture for two days. Afterharvest protein extracts were produced from leaf tissue of these plantsaccording to the method described under General Methods, Item 1a). Afterseparation of the proteins by means of denaturing polyacrylamide gelelectrophoreses 40 μg protein extract of each line was analysed by meansof Western Blot analysis using the antibody described under Examples,Item 10. As control samples, protein extracts from Arabidopsis plantsand from potato wildtype plants (cv Desiree) were also analysed. Plants,which exhibit an increased quantity of OK1 protein and a detectablequantity of A.t.-OK1 protein coding mRNA, were grown in the greenhouse.Starch, which was isolated from tubers of these plants, showed anincreased content of phosphate bonded covalently to the correspondingstarch.

13. Production of Transgenic Maize Plants, which Exhibit IncreasedActivity of an OK1 Protein

a) Manufacture of the Plasmid pUbi-A.t.-OK1

First the plasmid pIR96 was manufactured. The plasmid pIR96 was obtainedby cloning a synthetic piece of DNA consisting of the twooligonucleotides X1(TGCAGGCTGCAGAGCTCCTAGGCTCGAGTTAACACTAGTAAGCTTAATTAAGAT ATCATTTAC) andX2 (AATTGTAAATGATATCTTAATTAAGCTTACTAGTGTTAACTCGAGCCTAGGAGCTCTGCAGCCTGCA) into the vector pGSV71 excised with SdaI and MunI. Theplasmid obtained was excised with SdaI and the protruding 3′-ends weresmoothed with T4 DNA polymerase. The plasmid obtained was excised withSdaI, the protruding 3′-ends were smoothed with T4 DNA polymerase, and a197 base pair large HindIII/SphI fragment from pBinAR, smoothed with T4DNA polymerase (Höfgen and Willmitzer, 1990, Plant Science 66, 221-230),and containing the termination signal of the octopine synthase gene fromAgrobacterium tumefaciens, was inserted. The plasmid obtained wasdesignated as pIR96.

pGSV71 is a derivative of the plasmid pGSV7, which is derived from theintermediate vector pGSV1. pGSV1 constitutes a derivative of pGSC1700,the construction of which has been described by Cornelissen andVanderwiele (Nucleic Acid Research 17, (1989), 19-25). pGSV1 wasobtained from pGSC1700 by deletion of the carbenicillin resistance geneand deletion of the T-DNA sequences of the TL-DNA region of the plasmidpTiB6S3.

pGSV7 contains the replication origin of the plasmid pBR322 (Bolivar etal., Gene 2, (1977), 95-113) as well as the replication origin of thePseudomonas plasmid pVS1 (Itoh et al., Plasmid 11, (1984), 206). pGSV7also contains the selectable marker gene aadA, from the transposonTn1331 from Klebsiella pneumoniae, which gives resistance against theantibiotics spectinomycin and streptomycin (Tolmasky, Plasmid 24 (3),(1990), 218-226; Tolmasky and Crosa, Plasmid 29(1), (1993), 31-40). Theplasmid pGSV71 was obtained by cloning a chimeric bar gene between theborder regions of pGSV7. The chimeric bar gene contains the promotersequence of the cauliflower mosaic virus for initiating thetranscription (Odell et al., Nature 313, (1985), 180), the bar gene fromStreptomyces hygroscopicus (Thompson et al., Embo J. 6, (1987),2519-2523) and the 3′-untranslated area of the nopaline synthase gene ofthe T-DNA of pTiT37 for terminating the transcription andpolyadenylation. The bar gene provides tolerance against the herbicideglufosinate ammonium.

A 1986 base pair long fragment containing the promoter of thepolyubiquitin gene from maize (Genes from Maize (Gens aus Mais) (EMBLAcc.: 94464, Christensen et al., 1992, Plant Mol. Biol. 18: 675-689) wascloned as a PstI fragment into pBluescript II SK+. The plasmid obtainedwas designated as pSK-ubq.

The plasmid A.t.-OK1-pGEM was excised with the restriction enzymeBsp120I, the ends were smoothed with T4-DNA polymerase, and it wasre-excised with SacI. The DNA fragment coding the OK1 protein fromArabidopsis thaliana was cloned into the plasmid pSK-ubq, which wasexcised with SmaI and SacI. The plasmid obtained was designated aspSK-ubq-ok1.

A fragment was isolated from the plasmid pSK-ubq-ok1, which contains theubiquitin promoter from maize and the entire open reading frame for theA.t.-OK1 protein from Arabidopsis thaliana. For this purpose, theplasmid was excised with the restriction enzyme Asp718I, the ends werefilled with T4 DNA polymerase, and it was re-excised with SdaI. The 5799base pair large fragment obtained was cloned into the plasmid pIR96excised with EcoRV and PstI. The plasmid obtained from this cloning wasdesignated as pUbi-A.t.-OK1.

b) Transformation of Maize Plants

Maize plants were transformed with the plasmid pUbi-A.t.-OK1 using themethod described under General Methods, Item 17.

c) Analysis of the Transgenic Maize Plants and the Starch Synthesisedfrom these

Using Northern Blot analysis, plants could be identified, which exhibitan expression of mRNA, coding the A.t.-OK1 protein.

Maize plants, which exhibit a detectable amount of A.t.-OK1 proteincoding mRNA in comparison to corresponding wild type plants, were grownin the greenhouse. Single grains of these plants were harvested. Starch,isolated from these grains, showed an increased content of phosphatecovalently bonded to the respective starch in comparison to starch,which is isolated from grains of corresponding wild type plants.

14. Manufacture of Transgenic Wheat Plants, which Exhibit IncreasedActivity of an OK1 Protein

a) Manufacture of a Plasmid for the Transformation of Wheat Plants

pMCS5 (Mobitec, www.mobitec.de) was digested with BgIII and BamHI andre-inserted. The plasmid contained was designated as pML4.

The nos terminator from Agrobacterium tumefaciens (Depicker et al.,1982, Journal of Molecular and Applied Genetics 1: 561-573) wasamplified with the primers P9(ACTTCTgCAgCggCCgCgATCgTTCAAACATTTggCAATAAAgTTTC) and P10(TCTAAgCTTggCgCCgCTAgCAgATCTgATCTAgTAACATAgATgACACC) (25 cycles, 30 sec94° C., 30 sec 58° C., 30 sec 72° C.), digested with Hind/III and PstI,and cloned into the plasmid pML4 having been excised with the sameenzymes. The plasmid contained was designated as pML4-nos. A 1986 basepair long fragment containing the promoter of the polyubiquitin genefrom maize (Genbank Acc.: 94464, Christensen et al., 1992, Plant Mol.Biol. 18: 675-689) and the first intron of the same gene, shortenedthrough digestion by ClaI and re-insertion, were cloned into thisvector. The plasmid contained was designated as pML8.

The entire open reading frame of OK1 from Arabidopsis thaliana wascloned into the plasmid pML8. In order to this, the correspondingfragment with Bsp120/NotI was excised from A.t.-OK1-pGEM, and splicedinto the NotI site of pML8 in an “in sense” orientation.

A fragment for the transformation of wheat plants can be excised fromthe obtained vector pML8-A.t.-OK1 with the restriction enzymes AvrII andSwaI, which contains the promoter of the polyubiquitin gene from maize,the entire open reading frame of OK1 from Arabidopsis thaliana, and thenos terminator from Agrobacterium tumefaciens.

b) Transformation of Wheat Plants

Wheat plants of the Florida variety were transformed with a fragmentpurified from an agarose gel, which was excised with the restrictionenzymes AvrII and SwaI from the plasmid pML8-A.t.-OK1, and whichcontains the promoter of the polyubiquitin gene from maize, the entireopen reading frame of OK1 from Arabidopsis thaliana, and the nosterminator from Agrobacterium tumefaciens, and with the plasmid pGSV71using the biolistic method according to the method described by Beckeret al. (1994, Plant Journal 5, 299-307).

c) Analysis of the Transgenic Wheat Plants and the Starch Synthesisedfrom these

Starch was isolated from mature grains of the TO plants obtained fromthe transformation, and the content of phosphate covalently bonded tothe starch was determined. The phosphate content of the starch, whichwas isolated from individual grains, was clearly higher than in the caseof the starch, which was isolated from grains of corresponding wild typeplants.

The invention claimed is:
 1. A modified starch obtained from agenetically modified plant comprising one or more genetically modifiedplant cells that exhibit increased activity in at least one OK1 proteinin comparison to corresponding wild type plant cells that have not beengenetically modified, wherein said OK1 protein is: (a) encoded by anucleic acid molecule comprising the nucleotide sequence of SEQ ID NO:1, or the complementary sequence thereof; (b) encoded by a nucleic acidmolecule having at least 95% identity to the nucleotide sequence of SEQID NO: 1; (c) the amino acid sequence of SEQ ID NO: 2; (d) an amino acidsequence having at least 95% identity to the amino acid sequence of SEQID NO: 2; (e) encoded by a nucleic acid molecule coding a protein havingthe amino acid sequence of SEQ ID NO: 2; (f) encoded by a nucleic acidmolecule comprising the nucleotide sequence of SEQ ID NO: 3, or thecomplementary sequence thereof; (g) encoded by a nucleic acid moleculehaving at least 95% identity to the nucleotide sequence of SEQ ID NO: 3;(h) the amino acid sequence of SEQ ID NO: 4; (i) an amino acid sequencehaving at least 95% identity to the amino acid sequence of SEQ ID NO: 4;or (j) encoded by a nucleic acid molecule coding a protein having theamino acid sequence of SEQ ID NO: 4, and wherein the modified starch hasan increased proportion of starch phosphate bonded in the C-3 positioncompared with starch phosphate bonded in the C-6 position in comparisonto starch obtained from wild-type plants that have not been geneticallymodified.
 2. The modified starch of claim 1, wherein said OK1 protein isencoded by a nucleic acid molecule comprising the nucleotide sequence ofSEQ ID NO: 1, or the complementary sequence thereof.
 3. The modifiedstarch of claim 1, wherein said OK1 protein is encoded by a nucleic acidmolecule having at least 95% identity to the nucleotide sequence of SEQID NO:
 1. 4. The modified starch of claim 1, wherein said OK1 proteincomprises the amino acid sequence of SEQ ID NO:
 2. 5. The modifiedstarch of claim 1, wherein said OK1 protein comprises an amino acidsequence having at least 95% identity to the amino acid sequence of SEQID NO:
 2. 6. The modified starch of claim 1, wherein said OK1 protein isencoded by a nucleic acid molecule coding a protein having the aminoacid sequence of SEQ ID NO:
 2. 7. The modified starch of claim 1,wherein said OK1 protein is encoded by a nucleic acid moleculecomprising the nucleotide sequence of SEQ ID NO: 3, or the complementarysequence thereof.
 8. The modified starch of claim 1, wherein said OK1protein is encoded by a nucleic acid molecule having at least 95%identity to the nucleotide sequence of SEQ ID NO:
 3. 9. The modifiedstarch of claim 1, wherein said OK1 protein comprises the amino acidsequence of SEQ ID NO:
 4. 10. The modified starch of claim 1, whereinsaid OK1 protein comprises an amino acid sequence having at least 95%identity to the amino acid sequence of SEQ ID NO:
 4. 11. The modifiedstarch of claim 1, wherein said OK1 protein is encoded by a nucleic acidmolecule coding a protein having the amino acid sequence of SEQ ID NO:4.
 12. A method of manufacturing a modified starch according to claim 1,comprising extracting the starch from a genetically modified plantcomprising one or more genetically modified plant cells that exhibitincreased activity in at least one OK1 protein in comparison tocorresponding wild type plant cells that have not been geneticallymodified.
 13. A method of manufacturing a modified starch according toclaim 1, comprising extracting the starch from a harvestable plant partof a genetically modified plant comprising one or more geneticallymodified plant cells that exhibit increased activity in at least one OK1protein in comparison to corresponding wild type plant cells that havenot been genetically modified.
 14. A method of manufacturing a derivedstarch comprising deriving a modified starch according to claim
 1. 15. Amethod of manufacturing a modified starch comprising extracting thestarch from a genetically modified plant cell that exhibits increasedactivity in at least one OK1 protein in comparison to corresponding wildtype plant cells that have not been genetically modified, wherein saidOK1 protein is: (a) encoded by a nucleic acid molecule comprising thenucleotide sequence of SEQ ID NO: 1, or the complementary sequencethereof; (b) encoded by a nucleic acid molecule having at least 95%identity to the nucleotide sequence of SEQ ID NO: 1; (c) the amino acidsequence of SEQ ID NO: 2; (d) an amino acid sequence having at least 95%identity to the amino acid sequence of SEQ ID NO: 2; (e) encoded by anucleic acid molecule coding a protein having the amino acid sequence ofSEQ ID NO: 2; (f) encoded by a nucleic acid molecule comprising thenucleotide sequence of SEQ ID NO: 3, or the complementary sequencethereof; (g) encoded by a nucleic acid molecule having at least 95%identity to the nucleotide sequence of SEQ ID NO: 3; (h) the amino acidsequence of SEQ ID NO: 4; (i) an amino acid sequence having at least 95%identity to the amino acid sequence of SEQ ID NO: 4; or (j) encoded by anucleic acid molecule coding a protein having the amino acid sequence ofSEQ ID NO:
 4. 16. The method of claim 15, wherein said OK1 protein isencoded by a nucleic acid molecule comprising the nucleotide sequence ofSEQ ID NO: 1, or the complementary sequence thereof.
 17. The method ofclaim 15, wherein said OK1 protein is encoded by a nucleic acid moleculehaving at least 95% identity to the nucleotide sequence of SEQ ID NO: 1.18. The method of claim 15, wherein said OK1 protein comprises the aminoacid sequence of SEQ ID NO:
 2. 19. The method of claim 15, wherein saidOK1 protein comprises an amino acid sequence having at least 95%identity to the amino acid sequence of SEQ ID NO:
 2. 20. The method ofclaim 15, wherein said OK1 protein is encoded by a nucleic acid moleculecoding a protein having the amino acid sequence of SEQ ID NO:
 2. 21. Themethod of claim 15, wherein said OK1 protein is encoded by a nucleicacid molecule comprising the nucleotide sequence of SEQ ID NO: 3, or thecomplementary sequence thereof.
 22. The method of claim 15, wherein saidOK1 protein is encoded by a nucleic acid molecule having at least 95%identity to the nucleotide sequence of SEQ ID NO:
 3. 23. The method ofclaim 15, wherein said OK1 protein comprises the amino acid sequence ofSEQ ID NO:
 4. 24. The method of claim 15, wherein said OK1 proteincomprises an amino acid sequence having at least 95% identity to theamino acid sequence of SEQ ID NO:
 4. 25. The method of claim 15, whereinsaid OK1 protein is encoded by a nucleic acid molecule coding a proteinhaving the amino acid sequence of SEQ ID NO:
 4. 26. A method formanufacturing a flour comprising grinding plant cells that exhibitincreased activity in at least one OK1 protein in comparison tocorresponding wild type plant cells that have not been geneticallymodified, wherein said OK1 protein is: (a) encoded by a nucleic acidmolecule comprising the nucleotide sequence of SEQ ID NO: 1, or thecomplementary sequence thereof; (b) encoded by a nucleic acid moleculehaving at least 95% identity to the nucleotide sequence of SEQ ID NO: 1;(c) the amino acid sequence of SEQ ID NO: 2; (d) an amino acid sequencehaving at least 95% identity to the amino acid sequence of SEQ ID NO: 2;(e) encoded by a nucleic acid molecule coding a protein having the aminoacid sequence of SEQ ID NO: 2: (f) encoded by a nucleic acid moleculecomprising the nucleotide sequence of SEQ ID NO: 3, or the complementarysequence thereof; (g) encoded by a nucleic acid molecule having at least95% identity to the nucleotide sequence of SEQ ID NO: 3; (h) the aminoacid sequence of SEQ ID NO: 4; (i) an amino acid sequence having atleast 95% identity to the amino acid sequence of SEQ ID NO: 4; or (j)encoded by a nucleic acid molecule coding a protein having the aminoacid sequence of SEQ ID NO:
 4. 27. A flour comprising the starch ofclaim
 1. 28. A flour comprising the starch of claim
 2. 29. A flourcomprising the starch of claim
 3. 30. A flour comprising the starch ofclaim
 4. 31. A flour comprising the starch of claim
 5. 32. A flourcomprising the starch of claim
 6. 33. A flour comprising the starch ofclaim
 7. 34. A flour comprising the starch of claim
 8. 35. A flourcomprising the starch of claim
 9. 36. A flour comprising the starch ofclaim
 10. 37. A flour comprising the starch of claim
 11. 38. A methodfor manufacturing a flour comprising grinding plant parts that exhibitincreased activity in at least one OK1 protein in comparison tocorresponding wild type plant cells that have not been geneticallymodified, wherein said OK1 protein is: (a) encoded by a nucleic acidmolecule comprising the nucleotide sequence of SEQ ID NO: 1, or thecomplementary sequence thereof; (b) encoded by a nucleic acid moleculehaving at least 95% identity to the nucleotide sequence of SEQ ID NO: 1;(c) the amino acid sequence of SEQ ID NO: 2; (d) an amino acid sequencehaving at least 95% identity to the amino acid sequence of SEQ ID NO: 2;(e) encoded by a nucleic acid molecule coding a protein having the aminoacid sequence of SEQ ID NO: 2; (f) encoded by a nucleic acid moleculecomprising the nucleotide sequence of SEQ ID NO: 3, or the complementarysequence thereof; (g) encoded by a nucleic acid molecule having at least95% identity to the nucleotide sequence of SEQ ID NO: 3; (h) the aminoacid sequence of SEQ ID NO: 4; (i) an amino acid sequence having atleast 95% identity to the amino acid sequence of SEQ ID NO: 4; or (j)encoded by a nucleic acid molecule coding a protein having the aminoacid sequence of SEQ ID NO:
 4. 39. The method of claim 38, wherein saidOK1 protein is encoded by a nucleic acid molecule comprising thenucleotide sequence of SEQ ID NO: 1, or the complementary sequencethereof.
 40. The method of claim 38, wherein said OK1 protein is encodedby a nucleic acid molecule having at least 95% identity to thenucleotide sequence of SEQ ID NO:
 1. 41. The method of claim 38, whereinsaid OK1 protein comprises the amino acid sequence of SEQ ID NO:
 2. 42.The method of claim 38, wherein said OK1 protein comprises an amino acidsequence having at least 95% identity to the amino acid sequence of SEQID NO:
 2. 43. The method of claim 38, wherein said OK1 protein isencoded by a nucleic acid molecule coding a protein having the aminoacid sequence of SEQ ID NO:
 2. 44. The method of claim 38, wherein saidOK1 protein is encoded by a nucleic acid molecule comprising thenucleotide sequence of SEQ ID NO: 3, or the complementary sequencethereof.
 45. The method of claim 38, wherein said OK1 protein is encodedby a nucleic acid molecule having at least 95% identity to thenucleotide sequence of SEQ ID NO:
 3. 46. The method of claim 38, whereinsaid OK1 protein comprises the amino acid sequence of SEQ ID NO:
 4. 47.The method of claim 38, wherein said OK1 protein comprises an amino acidsequence having at least 95% identity to the amino acid sequence of SEQID NO:
 4. 48. The method of claim 38, wherein said OK1 protein isencoded by a nucleic acid molecule coding a protein having the aminoacid sequence of SEQ ID NO: 4.